A benzochromenonori-cidine alkaloid compound and application thereof

Abstract

The application belongs to the technical field of biological medicine, and relates to a benzochromenonoriwestine alkaloid compound and application thereof. A chemical structural formula is shown as formula I, wherein R is R' or LR', L is a connecting arm, and R' is R'' is an ether group, an imino group or an amido group. Research shows that the benzochromenonoriwestine alkaloid compound provided by the application has a GDI2 protein as an action target, and realizes inhibition of pancreatic tumor by degrading the GDI2 protein.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a benzoquinolone alkaloid compound and its applications. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Paraapoptosis is an atypical cell death process distinct from apoptosis, and its molecular mechanisms remain unclear. Previous studies have shown that activation of the MAPK / ERK and JNK / SAPK pathways can induce paraapoptosis, while AIP1 / Alix is ​​an endogenous inhibitor of paraapoptosis. Oxidative stress, overexpression of TAJ / TROY, and glucocorticoids can also induce or enhance paraapoptosis. Researching the mechanisms of paraapoptosis and designing related compounds is of great significance for overcoming the challenge of drug-resistant tumor cell lines resistant to apoptosis.

[0004] Many alkaloid small molecule compounds can induce significant paraapoptosis. In previous studies, the inventors discovered that 9,10-bisbenzyloxybenzo[a]quinoloxetine derivatives can induce typical paraapoptosis in tumor cells. However, the molecular targets that induce paraapoptosis in these compounds remain unknown, posing significant challenges to subsequent compound modification / bioactivity studies. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a benzoquinoloxetine alkaloid compound and its application. Studies have shown that the benzoquinoloxetine alkaloid compound provided by the present invention targets the GDI2 protein and inhibits pancreatic tumors by degrading the GDI2 protein.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] On the one hand, there is a benzoquinolone alkaloid compound, the chemical structure of which is shown in Formula I.

[0008]

[0009] Where R is R' or LR', L is the connecting arm, and R' is...

[0010] R” can be an ether group (-O-), an imino group (-NH-), or an amide group (-CONH-).

[0011] These compounds contain three asymmetric chiral carbon atoms at positions C-2, C-4, and C-11b, and exist as diastereomers and enantiomers, respectively. The asymmetric chiral carbon atom at position C-2, in some embodiments, has the chemical structure shown in Formula II or Formula III.

[0012]

[0013] The carbon atoms at positions C-2, C-4, and C-11b are asymmetric chiral carbon atoms, and in some embodiments, their chemical structures are shown in Formula IV or Formula V, respectively.

[0014]

[0015] In some embodiments, for

[0016] In some embodiments, for

[0017]

[0018] In some embodiments, the connecting arm is selected from

[0019] Wherein, n1 is an integer from 0 to 10, n2 is an integer from 1 to 5, n3 is an integer from 0 to 10, and n4 is an integer from 1 to 5. n1 is preferably an integer from 2 to 8 (e.g., 2, 3, 4, 5, 6, 7, 8). n2 is preferably an integer from 2 to 5 (e.g., 2, 3, 4, 5). n3 is preferably an integer from 3 to 5 (e.g., 3, 4, 5). n4 is an integer from 2 to 4 (e.g., 2, 3, 4).

[0020] In some embodiments, the compounds are selected from the following:

[0021] Rac-(2S,4S,11bR)-3-(4-((9,10-bis(3,5-dimethylbenzyloxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2-hydro-pyrido[2,1-a]isoquinoline-2-yl)methyl)-1-hydro-1,2,3-triazol-1-yl)-N-(2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxoisoindoline-4-yl)propionamide (93);

[0022] Rac-(2S,4S,11bR)-11-(4-((9,10-bis(3,5-dimethylbenzyloxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2-hydro-pyrido[2,1-a]isoquinoline-2-yl)methyl)-1-hydro-1,2,3-triazol-1-yl)-N-(2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxoisoindoline-4-yl)undecanoamide (94);

[0023] Rac-(2S,4S,11bR)-14-(4-(((9,10-bis(3,5-dimethylbenzyloxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2-hydro-pyrido[2,1-a]isoquinoline-2-yl)methyl)-1-hydro-1,2,3-triazol-1-yl)-N-(2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxoisoindoline-4-yl)-3,6,9,12-tetraoxotetradecanoamide (95);

[0024] Rac-(2S,4S,11bR)-5-((6-(4-((9,10-bis(3,5-dimethylbenzyloxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2-hydro-pyrido[2,1-a]isoquinoline-2-yl)methyl)-1-hydro-1,2,3-triazol-1-yl)n-hexyl)amino)-2-(2,6-dioxo-piperidin-3-yl)isoindole-1,3-dione(96);

[0025] Rac-(2S,4S,11bR)-5-((2-(2-(2-(4-((9,10-bis(3,5-dimethylbenzyloxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2-hydro-pyrido[2,1-a]isoquinoline-2-yl)methyl)-1-hydro-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxo-piperidin-3-yl)isoindole-1,3-dione(97);

[0026] Rac-(2S,4S,11bR)-N-(6-(4-((9,10-bis(3,5-dimethylbenzyloxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2-hydro-pyrido[2,1-a]isoquinoline-2-yl)methyl)-1-hydro-1,2,3-triazol-1-yl)n-hexyl)-2-((2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxoisoindoline-5-yl)amino)acetamide (98);

[0027] Rac-(2S,4S,11bR)-N-(2-(2-(2-(4-((9,10-bis(3,5-dimethylbenzyloxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2-hydro-pyrido[2,1-a]isoquinoline-2-yl)methyl)-1-hydro-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)-2-((2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)acetamide(99);

[0028] Rac-(2S,4S,11bR)-4-((6-(4-((9,10-bis(3,5-dimethylbenzyloxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2-hydro-pyrido[2,1-a]isoquinoline-2-yl)methyl)-1-hydro-1,2,3-triazol-1-yl)n-hexyl)amino)-2-(2,6-dioxo-piperidin-3-yl)isoindole-1,3-dione(100);

[0029] Rac-(2S,4S,11bR)-4-((2-(2-(2-(4-((9,10-bis(3,5-dimethylbenzyloxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2-hydro-pyrido[2,1-a]isoquinoline-2-yl)methyl)-1-hydro-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxo-piperidin-3-yl)isoindole-1,3-dione(101);

[0030] Rac-(2S,4S,11bR)-N-(6-(4-((9,10-bis(3,5-dimethylbenzyloxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2-hydro-pyrido[2,1-a]isoquinoline-2-yl)methyl)-1-hydro-1,2,3-triazol-1-yl)n-hexyl)-2-((2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)acetamide(102);

[0031] Rac-(2S,4S,11bR)-N-(2-(2-(2-(2-(4-((9,10-bis(3,5-dimethylbenzyloxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2-hydro-pyrido[2,1-a]isoquinoline-2-yl)methyl)-1-hydro-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)-2-((2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)acetamide(103);

[0032] Rac-(2S,4S,11bR)-4-((6-(4-((9,10-bis(3,5-dimethylbenzyloxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2-hydro-pyrido[2,1-a]isoquinoline-2-yl)methyl)-1-hydro-1,2,3-triazol-1-yl)n-hexyl)oxy)-2-(2,6-dioxo-piperidin-3-yl)isoindole-1,3-dione(104);

[0033] Rac-(2S,4S,11bR)-5-((6-(4-((9,10-bis(3,5-dimethylbenzyloxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2-hydro-pyrido[2,1-a]isoquinoline-2-yl)methyl)-1-hydro-1,2,3-triazol-1-yl)n-hexyl)oxy)-2-(2,6-dioxo-piperidin-3-yl)isoindole-1,3-dione(105);

[0034] Rac-(2S,4S,11bR)-4-(2-(2-(2-(2-(4-((9,10-bis(3,5-dimethylbenzyloxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2-hydro-pyrido[2,1-a]isoquinoline-2-yl)methyl)-1-hydro-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethoxy)-2-(2,6-dioxo-piperidin-3-yl)isoindole-1,3-dione(106);

[0035] Rac-(2S,4S,11bR)-5-(2-(2-(2-(4-((9,10-bis(3,5-dimethylbenzyloxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2-hydro-pyrido[2,1-a]isoquinoline-2-yl)methyl)-1-hydro-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethoxy)ethoxy)-2-(2,6-dioxo-piperidin-3-yl)isoindole-1,3-dione(107);

[0036] (R)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutanamide)-N-(6-(4-((Rac-(2S,4S,11bR)-9,10-bis(3,5-dimethylbenzyloxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2-hydro-pyrido[2,1-a]isoquinoline-2-yl)methyl)-1H-1,2,3-triazol-1-yl)n-hexyl)-4-methylpentanamide (108);

[0037] (R)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutanamide)-N-(2-(2-(2-(2-(4-(((2S,4S,11bR)-9,10-bis(3,5-dimethylbenzyloxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2-hydro-pyrido[2,1-a]isoquinoline-2-yl)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)-4-methylpentanamide (109);

[0038] (2S,4R)-1-((S)-2-(6-(4-((Rac-(2S,4S,11bR)-9,10-bis(3,5-dimethylbenzyloxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2-hydro-pyrido[2,1-a]isoquinoline-2-yl)methyl)-1H-1,2,3-triazol-1-yl)hexanoyl)-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide(110);

[0039] (2S,4R)-1-((S)-14-(4-((Rac-(2S,4S,11bR)-9,10-bis(3,5-dimethylbenzyloxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2-hydro-pyrido[2,1-a]isoquinoline-2-yl)methyl)-1H-1,2,3-triazol-1-yl)-2-(tert-butyl)-4-oxo-6,9,12-trioxo-3-tetradecanoamide)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (111).

[0040] On the other hand, a pharmaceutical preparation comprising the aforementioned benzoquinol alkaloid compound or its pharmaceutically acceptable salt, solvate, hydrate, prodrug compound, polymorph, or corresponding active metabolite.

[0041] In some embodiments, the pharmaceutically acceptable salt includes salts formed by the above-described compounds with inorganic acids, such as hydrochloric acid, sulfuric acid, nitric acid, or hydrobromic acid, and salts formed with organic acids, such as methanesulfonic acid, toluenesulfonic acid, or trifluoroacetic acid.

[0042] In some embodiments, the dosage form of the pharmaceutical preparation is a tablet, capsule, lozenge, sugar-coated pill, granule, powder, solution, emulsion, suspension, dispersion, syrup, gel, aerosol, or other special dosage form.

[0043] In some embodiments, the pharmaceutical formulation further includes pharmaceutical excipients. The pharmaceutical excipients include excipients or pharmaceutical carriers.

[0044] Thirdly, the application of one of the above-mentioned benzoquinol alkaloid compounds or pharmaceutical preparations in the preparation of GDI2 protein degrading agents.

[0045] Fourthly, the application of one of the above-mentioned benzoquinol alkaloid compounds or pharmaceutical preparations in the preparation of antitumor drugs.

[0046] In some embodiments, the antitumor drug is an antipancreatic cancer drug.

[0047] Fifthly, an application of GDI2 protein as a drug target in the preparation of antitumor drugs.

[0048] In some embodiments, the drug target of the above-mentioned benzoquinol alkaloid compound or pharmaceutical preparation is the GDI2 protein.

[0049] Sixthly, a biotinylated benzoquinolone alkaloid probe, the chemical structure of which is shown below:

[0050]

[0051] The biotinylated benzoquinolixidine alkaloid probes provided by this invention can identify molecular targets and perform target separation using the classic method of binding of compounds to target proteins based on their affinity. It has been found that the target of the benzoquinolixidine alkaloid compounds provided by this invention is the Rab-GDI2 protein, and further research shows that the target is the GDI2 protein.

[0052] The beneficial effects of this invention are as follows:

[0053] 1. This invention is the first to use biotinylated benzoquinoloxetine alkaloid probes to identify Guanyl Nucleotide Dissociation Inhibitor β (GDI2) as a key target protein in the process of paraapoptosis induced by benzoquinoloxetine alkaloids in tumor cells.

[0054] 2. Based on the discovered target protein, this invention further provides that benzoquinolone alkaloids have a good inhibitory effect on pancreatic cancer cells AsPC-1. Studies have shown that they can significantly reduce the level of the target GDI2 protein in pancreatic cancer cells AsPC-1.

[0055] 3. The benzoquinoloxetine alkaloids provided by this invention can induce the degradation of intracellular GDI2 protein. Western blot analysis showed that some benzoquinoloxetine alkaloids can degrade GDI2 protein by up to 40%.

[0056] 4. The benzoquinoloxetine alkaloids provided by this invention can effectively inhibit the growth of pancreatic tumor AsPC-1 in vivo at the animal level. Some benzoquinoloxetine alkaloids (e.g., compound 109) can significantly inhibit tumor growth with lower dosage. Attached Figure Description

[0057] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0058] Figure 1 The following are examples from Example 9 of this invention: (A) the spontaneous vacuolization phenomenon in PC-3 cells after downregulation of GDI2 protein expression; (B) the effect of GDI2 siRNA on GDI2 protein expression in PC-3 cells detected by Western blotting; and (C) the effect of alkaloids on the proliferation activity of d-GDI2 PC-3 cells.

[0059] Figure 2 This is a diagram showing the results of GDI2 degradation induced by PROTACs-type small molecules in Example 6 of this invention;

[0060] Figure 3 The image shows the tumor volume (A) and weight (B) results in Example 7 of this invention. Detailed Implementation

[0061] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0062] Example 1: Synthesis of key propyne-based intermediate compounds

[0063] Synthesis of intermediate compounds 1 and 4:

[0064]

[0065] First, following the method described in the literature (J.Med.Chem.2016,59,5063-5076), a racemic compound 1-1 was synthesized through an 8-step reaction. Then, bright magnesium strips (864 mg, 36 mmol, 3.6 eq) were cut into small pieces, and a catalytic amount of Hg₂Cl₂ (60 mg, 0.22 mmol, 0.022 eq) was placed in a round-bottom flask, and 5 mL of anhydrous diethyl ether was added. At 0 °C, an anhydrous diethyl ether solution of 3-bromopropyne (1.2 g, 10 mmol, 1 eq) was added dropwise, and the reaction was allowed to proceed for 1 h. Compound 1-1 (1.25 g, 2.6 mmol, 0.25 eq) was dissolved in THF (50 mL), cooled to 0 °C, and then added to the aforementioned 3-bromopropyne Grignard reagent (1.0 M solution in Et₂O, 5 mL). After the reaction was brought to room temperature and stirred for 1 h, saturated NH₄Cl (15 mL) was added. Extracted three times with CH₂Cl₂ (3 × 50 mL), evaporated to dryness, and separated by column chromatography to obtain intermediate compound 1 (922 mg, 73%) and compound 4 (38 mg, 3%), both white solids. Compound 1: Colorless crystal, mp. 44-46℃. 1 H NMR (400MHz, Methanol-d4) δ7.06(d,J=8.8Hz,4H),6.99-6.91(m,2H),6.84(s,1H),6.78(s,1H),5.02(d,J=4.0Hz,4H),4.14(d,J=9.7Hz,1H),3.29-

[0066] 3.19(m,1H),3.05-2.92(m,2H),2.87-2.69(m,2H),2.41-2.23(m,15H),2.16-2.03(m,2H),1.79-1.60(m,2H),1.36(d,J=7.1Hz,3H). 13 C NMR(101MHz,MeOD)δ147.7,147.0,137.6,137.6,137.2,137.2,128.9,128.9,127.4,125.3,125.1,11 4.9,113.5,79.9,71.6,71.1,71.0,70.1,55.0,49.5,40.7,37.8,33.9,28.0,20.0,20.0,13.6.ESI-MS m / z 524[M+H] + HR-EI-MS: m / z 524.3153 [M+H] + ,(calcd for C 35 H 42 NO3 +,524.3159).Compound 4.Yellowoil. 1 H NMR(400MHz,Methanol-d4)δ7.01(d,J=5.1Hz,4H),6.91(d,J=5.6Hz,2H),6.80(s,1 H),6.73(s,1H),4.96(d,J=3.7Hz,4H),3.36-3.31(m,1H),3.29-3.22(m,1H),2.96-

[0067] 2.83(m,1H),2.67(dt,J=12.2,4.6Hz,1H),2.59(dd,J=22.8,2.8Hz,1H),2.53-2.38(m,3H),2.25(d ,J=3.2Hz,14H),1.86(dt,J=13.2,2.8Hz,1H),1.54(td,J=12.7,5.9Hz,2H),1.17(d,J=6.2Hz,3H). 13 C NMR(101MHz,MeOD)δ147.9,147.0,137.6,137.6,137.2,137.2,130.1,128.9,128.9,127.6,125.2,125.0 ,114.6,112.9,79.9,71.6,71.2,70.9,69.1,59.5,54.8,43.9,43.8,41.4,28.4,27.8,20.0,19.0.ESI-MS m / z 524[M+H] + HR-EI-MS: m / z 524.3162 [M+H] + ,(calcd for C 35 H 42 NO3 + ,524.3159).

[0068] Synthesis of intermediate compounds 2 and 3:

[0069]

[0070] First, compounds 2-3 were synthesized with reference to the literature (J.Med.Chem.2016,59,5063-5076).

[0071] Compound 2-3 (800 mg, 2 mmol, 1 eq) was dissolved in 2 mL of CH2Cl2. NBS (391 mg, 2.2 mmol, 1.1 eq) was slowly added at 0 °C, and the reaction was carried out for 1 h. After adding 3 mL of 30% NaOH solution, the mixture was stirred at room temperature for 1 h, separated, and extracted with CH2Cl2 (3 × 20 mL). The organic phase was dried, filtered, and evaporated to dryness before column chromatography to obtain the corresponding imine product 2-2 (380 mg, 48%).

[0072] Pale yellow solid. 1 H NMR(400MHz,Chloroform-d)δ8.18(d,J=2.3Hz,1H),7.07(s,4H),7.02-6.92(m,2H),6.89(s,1H),6.74(s,1H), 5.10(d,J=14.4Hz,4H),3.70(td,J=8.6,8.1,2.3Hz,4H),2.64(t,J=7.9Hz,2H),2.31(d,J=3.3Hz,12H).ESI-MS m / z 400[M+H] + .

[0073] Two corresponding thiourea chiral catalysts, which are enantiomers, were synthesized according to the procedure described in reference (J. Am. Chem. Soc. 2013, 135, 1891-1894). Following similar Aza-Diels-Alder reaction conditions, compound 2-2 (640 mg, 1.6 mmol, 1 eq) was dissolved in 16 mL of 0.015 M AcOH toluene solution (0.24 mmol, 0.15 eq). After cooling to 0 °C, the corresponding thiourea catalyst (112 mg, 0.24 mmol, 0.15 eq) and freshly prepared methylbutenone (236 μL, 2.43 mmol, 1.5 eq) were added. After stirring at 4 °C for 2 days, the reaction was directly separated by column chromatography to obtain the corresponding single-configuration products. By using thiourea catalysts with different configurations, enantiomers 2-1 (500 mg, 65%) or 3-1 (535 mg, 69%) were obtained, respectively. Compound 2-1. Colorless crystal. 1¹H NMR (400 MHz, Chloroform-d) δ 7.06 (d, J=6.4 Hz, 4H), 6.94 (s, 2H), 6.71 (s, 1H), 6.63 (s, 1H), 5.03 (d, J=11.0 Hz, 4H), 4.12 (dd, J=11.4, 3.8 Hz, 1H), 3.57 (p, J=6.5 Hz, 1H), 3.09 (dt, J=13.0, 6.4 Hz, 1H), 3.00-2.88 (m, 1H), 2.88-2.74 (m, 3H), 2.61 (d, J=12.7 Hz, 1H), 2.47 (dd, J=14.1, 11.0 Hz, 1H), 2.31 (s, 12H), 2.23 (d, J=13.4 Hz, 1H), 1.12 (d, J=7.0 Hz, 3H). 13 ¹³C NMR (101 MHz, CDCl₃) δ 209.3, 148.0, 147.4, 138.0, 137.2, 137.1, 130.0, 129.4, 129.4, 127.1, 125.2, 125.1, 115.2, 112.9, 71.8, 71.4, 57.6, 53.9, 47.4, 47.0, 46.3, 29.5, 21.3, 14.9. ESI-MS m / z 484 [M+H] + , HR-EI-MS: m / z 484.2852 [M+H] + , (calcd for C 32 H 38 NO₃ + , 484.2846).

[0074] t R =10.91 min (Chiralpak AS-H column, 20% isopropanol / 80% hexanes, 1 mL / min). [α] 20 D =+19.00°, c=0.5

[0075] mg / mL in chloroform. Compound 3-1. Colorless crystal. 1 ¹H NMR (400 MHz, Chloroform-d) δ 7.06 (d, J=6.8 Hz,

[0076] 4H),6.93(d,J=3.4Hz,2H),6.70(s,1H),6.63(s,1H),5.03(d,J=11.7Hz,4H),4 .12(dd,J=11.2,3.8Hz,1H),3.56(td,J=6.7,2.1Hz,1H),3.10(dd,J=10.5,5.5H z,1H),3.02-2.72(m,4H),2.61(ddd,J=14.1,3.6,2.1Hz,1H),2.46(dd,J=14.1, 11.2Hz,1H),2.30(s,12H),2.23(dt,J=13.6,2.1Hz,1H),1.12(d,J=6.8Hz,3H). 13 C NMR (101MHz, CDCl3) δ209.3,148.0,147.4,138.0,137.2,137.1,130.0,129.4,129.4,127.1,1 25.2,125.1,115.2,112.9,71.8,71.4,57.6,53.9,47.4,47.0,46.3,29.5,21.3,14.9.ESI-MS m / z 484[M+H] + HR-EI-MS: m / z 484.2851 [M+H] + ,(calcd for C 32 H 38 NO3 + ,484.2846).t R =5.51min (Chiralpak AS-H column, 20% isopropanol / 80% hexanes, 1mL / min).[α] 20 D =-20.20°,c=0.5mg / mL in chloroform.

[0077] The synthesized compounds 2-1 and 3-1 react with propargyl Grignard reagents to give corresponding single-configuration alkynyl derivatives 2 and 3. Compound 2, colorless crystal, mp. 45-46℃. 11H NMR (400 MHz, Chloroform-d) δ 7.06 (s, 4H), 6.93 (s, 2H), 6.79 (s, 1H), 6.67 (s, 1H), 5.07-4.97 (m, 4H), 4.09 (d, J=9.7 Hz, 1H), 3.30-3.15 (m, 1H), 3.04-2.86 (m, 2H), 2.86-2.74 (m, 1H), 2.68-2.56 (m, 1H), 2.30 (s, 14H), 2.14 (t, J=2.6 Hz, 1H), 2.09 (dt, J=13.8, 2.8 Hz, 1H), 1.98-1.92 (m, 1H), 1.72 (dt, J=14.2, 2.6 Hz, 1H), 1.57-1.50 (m, 1H), 1.29 (d, J=7.0 Hz, 3H). 13 13C NMR (101 MHz, CDCl3) δ 147.9, 147.3, 138.0, 137.9, 137.3, 137.2, 129.4, 129.4, 127.8, 125.3, 125.1, 115.2, 113.5, 79.8, 72.4, 72.1, 71.4, 70.5, 54.7, 50.1, 48.3, 42.6, 40.2, 34.8, 28.8, 21.3, 13.9. ESI-MS m / z 524 [M+H] + , HR-EI-MS: m / z 524.3159 [M+H] + , (calcd for C 35 H 42 NO3 + , 524.3159). t R = 11.33 min (Chiralpak AS-H column, 5% isopropanol / 95% hexanes, 1 mL / min). [α] 20 D = -22.90°, c = 0.5 mg / mL in methanol. Compound 3, Colorless crystal, mp. 45-47 °C. 1H NMR(400MHz,Chloroform-d)δ7.06(s,4H),6.93(s,2H),6.79(s,1H),6.67(s,1H),5.07-4 .97(m,4H),4.09(d,J=9.7Hz,1H),3.30-3.15(m,1H),3.04-2.86(m,2H),2.86-2.74(m,1H ),2.68-2.56(m,1H),2.30(s,14H),2.14(t,J=2.6Hz,1H),2.09(dt,J=13.8,2.8Hz,1H),1 .98-1.92(m,1H),1.72(dt,J=14.2,2.6Hz,1H),1.57-1.50(m,1H),1.29(d,J=7.0Hz,3H). 13 C NMR (101MHz, CDCl3) δ147.7,147.2,137.9,137.9,137.4,137.3,129.4,129.3,128.2,125.3,125.1,11 5.3,113.6,80.0,72.2,72.1,71.5,70.7,54.3,49.7,48.2,42.8,40.5,34.9,29.2,21.3,13.9.ESI-MS m / z 524[M+H] + ,HR-EI-MS:m / z

[0078] 524.3160 ​​[M+H] + ,(calcd for C 35 H 42 NO3 + ,524.3159).t R =8.38min(Chiralpak AS-H column,5%

[0079] isopropanol / 95% hexanes,1mL / min).[α] 20 D =+15.40°,c=0.5mg / mL inmethanol.

[0080] Example 2: Synthesis of biotinylated benzoquinoloxetine alkaloid probe (compound 5)

[0081] Compound 1 (30 mg, 0.057 mmol, 1 eq) and its corresponding biotinylated azide, Biotin-PEG2-N3 (0.086 mmol, 34 mg, 1.5 eq), were dissolved in 2 mL of water. tIn a 1:1 mixture of BuOH / H₂O, a small amount of TBTA and 400 μL of sodium ascorbate (1 M) were added, followed by 150 μL of CuSO₄ (0.5 M), and the mixture was reacted at room temperature for 1 h. The reaction solution was extracted three times with CH₂Cl₂ (3 × 10 mL), evaporated to dryness, and then separated by column chromatography to obtain compound 5 (28 mg, 53%). Yellow crystal, mp. 86-88℃. 1 H NMR (400MHz, Methanol-d4) δ7.84(s,1H),7.06(s,4H),6.96(s,2H),6.84(s,1H),6.79(s,1H),5.01(d,J=4.0Hz,4H),4.56(t,J=5.1H z,2H),4.45(dd,J=7.9,5.0Hz,1H),4.25(dd,J=7.9,4.5Hz,1H),4.17(d,J=10.7Hz,1H),3.89(t,J=5.7Hz,2H),3.58(dt,J=5.7,3.5H z,2H),3.51(dd,J=5.7,3.3Hz,2H),3.46(t,J=5.6Hz,2H),3.32(s,2H),3.23(s,1H),3.15(dt,J=10.4,5.7Hz,1H),3.01-2.64(m,8H) ,2.29(s,12H),2.18(t,J=7.4Hz,2H),2.06-1.93(m,2H),1.62(pd,J=15.5,8.3Hz,6H),1.41(q,J=7.3Hz,2H),1.35(d,J=6.8Hz,3H). 13 C NMR(101MHz,MeOD)δ174.7,164.7,147.7,147.1,143.0,137.6,137.2,137 .2,130.7,129.0,128.9,127.5,125.3,125.1,124.6,114.8,113.3,71.6,7 1.0,70.4,70.0,69.8,69.2,69.0,61.9,60.2,55.6,55.0,49.9,49.6,41. 3,40.0,39.6,38.9,38.3,35.3,28.4,28.1,25.4,20.1,20.0,13.7.ESI-MS m / z 924[M+H] + HR-EI-MS: m / z 924.5070 [M+H] + ,(calcd for C 51 H 70 N7O7S +,924.5052).

[0082] Example 3: Synthesis of PROTACs-type azide fragments:

[0083]

[0084] Synthesis of connecting arms L1-L6:

[0085]

[0086] Synthesis of L1. The synthetic route was followed according to J. Am. Chem. Soc. 2010, 132, 656-666. PBr3 (10.2 g, 37.7 mmol, 0.8 eq) was added dropwise to polyethylene glycol (9.13 g, 47.1 mmol, 1 eq) at 0 °C. After returning to room temperature, the mixture was heated to 50-60 °C and stirred for 12 h. Extraction was performed with DCM, followed by washing with saturated NaHCO3 solution and water, drying, and column chromatography to obtain L1-2 (8.4 g, 56%). 1 ¹H NMR (400MHz, Chloroform-d) δ 3.82 (t, J = 6.3Hz, 4H), 3.75–3.64 (m, 8H), 3.48 (t, J = 6.3Hz, 4H). L1-2 (8.4 g, 26.4 mmol, 1 eq) was dissolved in 40 mL of anhydrous DMF, NaN3 was added, and the mixture was stirred at 60 °C for 3 h. The reaction was then terminated by adding water. The product was extracted with DCM (3 × 20 mL), dried, and then subjected to column chromatography to obtain the corresponding product L1-2 (5.7 g, 88%). 1 ¹H NMR (400MHz, Chloroform-d) δ 3.75–3.61 (m, 12H), 3.39 (t, J = 5.1 Hz, 4H). Intermediate L1-2 (1.25 g, 5 mmol, 1 eq) was dissolved in a solution of Et₂O / (1N)HCl / THF (6 / 1.2 / 6 mL). 7 mL of PPh₃ (1.31 g, 5 mmol, 1 eq) in diethyl ether was added dropwise. After stirring at room temperature for 5 h, the mixture was separated into EtOAc and water (100:100 mL). The aqueous phase was adjusted to pH 14 with NaOH and then extracted with DCM (3 × 50 mL). After drying and evaporation, column chromatography (DCM / MeOH / NH₄OH) was performed to obtain product L1 (360 mg, 33%). 1 H NMR (400MHz, Chloroform-d) δ3.80-3.59(m,10H),3.51(t,J=5.2Hz,2H),3.39(t,J=5.1Hz,2H),2.87(t,J=5.2Hz,2H). 13C NMR (101MHz, CDCl3) δ73.5,70.8,70.7,70.7,70.3,70.1,50.7,41.8.

[0087]

[0088] Synthesis of L2. Using 1,6-hexanediol as the starting material, the linker arm L2 was synthesized following the synthetic steps of L1. L2-3, 1 HNMR(400MHz,Chloroform-d)δ3.28(t,J=6.8Hz,2H),1.62(p,J=7.0Hz,2H),1.49-1.34(m,2H).L2, 1 H NMR (400MHz, Chloroform-d) δ3.27 (t, J = 6.9 Hz, 2H), 2.69 (t, J = 6.9 Hz, 2H), 1.61 (p, J = 6.9 Hz, 2H), 1.51-1.31 (m, 6H).

[0089]

[0090] Synthesis of L3. First, L3-2 was synthesized according to Chem. Comm. 2017, 53, 9024-9027. Intermediate L3-2 (5.12 g, 20 mmol, 1 eq) was dissolved in 30 mL of anhydrous DMF, and NaN3 (2.6 g, 40 mmol, 2 eq) was added. The mixture was stirred at room temperature for 6 h, and the reaction was terminated by adding water. The mixture was extracted with DCM (3 × 20 mL), dried, and then subjected to column chromatography to obtain the corresponding product L1-2 (3.3 g, 76%). 1 ¹H NMR (400 MHz, Chloroform-d) δ 3.76–3.66 (m, 12H), 3.65–3.58 (m, 2H), 3.40 (t, J = 5.1 Hz, 2H). PPh₃ (3.93 g, 15 mmol, 1.5 eq) and CBr₄ (4.96 g, 15 mmol, 1.5 eq) were dissolved in 40 mL of EtOAc. After stirring for 10 min, 10 mL of an EtOAc solution of L₃⁻₃ (2.2 g, 10 mmol, 1 eq) was added. After stirring for two hours, the mixture was filtered through diatomaceous earth, evaporated to dryness, and subjected to column chromatography to obtain product L₃ (2.4 g, 85%). 1 H NMR (400MHz, Chloroform-d) δ3.82(t,J=6.4Hz,2H),3.68(s,10H),3.48(t,J=6.4Hz,2H),3.40(t,J=5.2Hz,2H).

[0091]

[0092] Synthesis of L4. Starting with L4-1, the connecting arm L4 was synthesized following the steps in L3. L4-2, 1 H NMR(400MHz,Chloroform-d)δ3.65(t,J=6.5Hz,2H),3.28(t,J=6.8Hz,2H),1.64-1.54(m,4H),1.48-1.33(m,4H).L4, 1 H NMR (400MHz, Chloroform-d) δ3.42(t,J=6.7Hz,2H),3.28(t,J=6.9Hz,2H),1.88(p,J=6.8Hz,2H),1.62(p,J=7.0Hz,2H),1.54-1.34(m,4H).

[0093]

[0094] Synthesis of L5. Fmoc-protected glycine L5-1 (115 mg, 0.39 mmol, 1.1 eq) and L2 (50 mg, 0.35 mmol, 1 eq) were dissolved in 2 mL of DMF. HATU (200 mg, 0.53 mmol, 1.5 eq) and DIEA (123 μL, 0.7 mmol, 2 eq) were added sequentially, and the reaction was allowed to proceed for 10 min. The reaction was terminated with water and extracted with DCM (3 × 20 mL). After drying and separation by column chromatography, intermediate L5-2 (150 mg, quantitive) was obtained. 1 ¹H NMR (400MHz, Chloroform-d) δ 7.77 (d, J = 7.5Hz, 2H), 7.59 (d, J = 7.3Hz, 2H), 7.41 (t, J = 7.2Hz, 2H), 7.32 (t, J = 7.5Hz, 2H), 5.85 (s, 1H), 5.35 (s, 1H), 4.46 (d, J = 6.5Hz, 2H), 4.23 (t, J = 6.8Hz, 1H), 3.90–3.75 (m, 2H), 3.40–3.16 (m, 4H), 1.63–1.52 (m, 4H), 1.45–1.26 (m, 4H). Compound L5-2 (150 mg, 0.35 mmol, 1 eq) was dissolved in 1 mL of anhydrous CH₂Cl₂, and 1 mL of... After stirring Et2N overnight, the product was directly evaporated to dryness and subjected to column chromatography to obtain product L5 (65 mg, 93%). ¹H NMR (400 MHz, Chloroform-d) δ 3.36 (s, 2H), 3.34–3.18 (m, 4H), 1.66–1.49 (m, 4H), 1.47–1.31 (m, 4H).

[0095]

[0096] Synthesis of L6. Following the synthetic route of L5, the connecting arm L6 was prepared. L6-1, 1 H NMR(400MHz,Chloroform-d)δ7.77(d,J=7.2Hz,2H),7.60(d,J=7.2Hz,2H),7.41(t,J=7.4Hz,2H),7.32(t,J=6.9Hz,2H), 6.46(s,1H),5.58(s,1H),4.44(d,J=6.8Hz,2H),4.23(t,J=7.2Hz,1H),3.88(d,J=6.0Hz,2H),3.72-3.53(m,10H),3.53-

[0097] 3.45(m,2H),3.36(t,J=5.1Hz,2H).L6, 1 H NMR (400MHz, Chloroform-d) δ3.73-3.62(m,8H),3.59(t,J=5.1Hz,2H),3.53-3.47(m,2H),3.40(t,J=5.3Hz,2H).

[0098]

[0099] Synthesis of L7. Jones' reagent was prepared by dissolving CrO3 (300 mg, 3 mmol, 3 eq) in 10 mL of 1.5 M H2SO4. This reagent was then added dropwise to an acetone solution of L3-3 (219 mg, 1 mmol, 1 eq) at 0 °C and stirred overnight at room temperature. The reaction was terminated by adding 16 mL of isopropanol. The mixture was extracted with DCM (3 × 20 mL), dried, and separated by column chromatography to obtain compound L7 (104 mg, 44%). 1 H NMR (400MHz, Chloroform-d) δ4.21-4.14 (m, 2H), 3.90-3.57 (m, 10H), 3.41 (t, J = 4.6Hz, 2H).

[0100]

[0101] Synthesis of L8. NaH (60%, 1.1 g, 27.4 mmol, 6 eq) was added to 40 mL of THF, followed by L3-3 (1 g, 4.6 mmol, 1 eq), and 20 mL of bromoacetic acid THF solution (1.27 g, 9.2 mmol, 2 eq) was slowly added dropwise. The mixture was stirred overnight. Excess NaH was carefully neutralized with cold water, and the THF was evaporated to dryness. 50 mL of water was added, and the reaction solution was neutralized to pH < 5 with 1 M HCl. After extraction with DCM (3 × 20 mL), the organic phases were combined, dried, evaporated to dryness, and subjected to column chromatography to obtain the product L8 (750 mg, 65%). ESI-MS m / z 251 [MH] + .

[0102] Synthesis of thalidomide derivatives S80-S91:

[0103]

[0104] Synthesis of compounds S80 and S81. S80-1 (1.66 g, 10 mmol, 3 eq), S80-2 (1.64 g, 10 mmol, 1 eq) and AcONa (984 mg, 12 mmol, 1.2 eq) were dissolved in 30 mL of AcOH, heated to 140 °C and refluxed for 12 h, then directly evaporated to dryness and subjected to column chromatography to obtain product S80-3 (1.5 g, 54%). 1 H NMR (400MHz, DMSO-d6) δ8.08-7.88(m,1H),7.88-7.58(m,2H),5.17(dd,J=12.9,5.4 Hz,1H),3.02-2.80(m,1H),2.72-2.57(m,1H),2.56-2.50(m,1H),2.20-1.97(m,1H).

[0105] S80-3 (131 mg, 0.5 mmol, 1 eq), L2 (71 mg, 0.5 mmol, 1 eq), and DIEA (178 μL, 1 mmol, 2 eq) were dissolved in 2 mL of DMF, and the mixture was heated to 90 °C and reacted for 12 h until the starting material disappeared. 20 mL of EtOAc was added and the mixture was washed with water. The organic phase was dried, evaporated to dryness, and the product S-80 (103 mg, 52%) was separated by column chromatography. 1H NMR(400MHz,Chloroform-d)δ8.28(s,1H),7.56(t,J=7.8Hz,1H),7.25(d,J=7.1Hz,1H),6.82(d,J=8.4Hz,1H),6.67(t,J=5.8Hz,1H),6.54(t,J=6.1Hz,1H) ,4.95(dd,J=12.2,5.4Hz,1H),3.96(d,J=6.0Hz,2H),3.44-3.08(m,4H),2.98 -2.71(m,3H),2.20-2.08(m,1H),1.61-1.41(m,4H),1.39-1.25(m,4H).ESI-MS m / z 399[M+H] + .

[0106] S-81 was prepared using the same procedures as S-80. 1 H NMR(400MHz,Chloroform-d)δ7.97(s,1H),7.49(t,J=7.9Hz,1H),7.11(d,J=7.0Hz,1H),6.93(d,J=8.6Hz,1H),6.49(s,1H),4.91(dd ,J=12.2,5.5Hz,1H),3.86-3.54(m,12H),3.47(t,J=5.4Hz,2H),3.38(t,J=5.1Hz,2H),3.02-2.67(m,3H),2.17-2.10(m,1H).ESI-MS m / z 475[M+H] + .

[0107]

[0108] Synthesis of compounds S82 and S83. S80-3 (550 mg, 2 mmol, 1 eq), glycine tert-butyl ester (260 mg, 2 mmol, 1 eq), and DIEA (700 μL, 4 mmol, 2 eq) were dissolved in 20 mL of DMSO and reacted at 90 °C for 1 day. EtOAc and water were added for liquid-liquid separation and washing with water. The organic phase was dried and evaporated to dryness before column chromatography to obtain product S83-1 (400 mg, 50%). 1¹H NMR (400MHz, Chloroform-d) δ 7.51 (t, J = 7.8Hz, 1H), 7.16 (d, J = 7.2Hz, 1H), 6.76 (d, J = 8.4Hz, 1H), 6.71 (t, J = 5.6Hz, 1H), 4.93 (dd, J = 12.3, 5.3Hz, 1H), 3.95 (d, J = 5.5Hz, 2H), 2.96–2.68 (m, 3H), 2.18–2.10 (m, 1H), 1.50 (s, 9H). S83-1 (390mg, 1mmol, 1eq) was directly dissolved in 18mL of DCM, and 1.8mL of TFA was added dropwise at 0℃. After reacting for 10h at room temperature, the solvent was directly evaporated to obtain product S82-2 (310mg, 93%). 1 H NMR (400MHz, DMSO-d6) δ12.88(s,1H),11.10(s,1H),7.59(t,J=7.8Hz,1H),7.08(d,J=7.1Hz,1H),6.99(d,J=8.7Hz,1H),6.95-6.74(m ,1H),5.07(dd,J=13.0,5.3Hz,1H),4.11(d,J=4.4Hz,2H),2.89(ddd,J=19.1,14.1,5.4Hz,1H),2.67-2.52(m,2H),2.17-1.89(m,1H).

[0109] S83-2 (66 mg, 0.2 mmol, 1 eq) and L1 (52 mg, 0.22 mmol, 1.1 eq) were dissolved in 1 mL of DMF. HATU (114 mg, 0.3 mmol, 1.5 eq) and DIEA (73 μL, 0.4 mmol, 2 eq) were then added, and the mixture was reacted for 10 min. The reaction was terminated by adding water and extracted with DCM (3 × 20 mL). After drying and separation by column chromatography, intermediate S-83 (85 mg, 80%) was obtained. 1 H NMR(400MHz,Chloroform-d)δ8.21(s,1H),7.54(t,J=7.8Hz,1H),7.22(d,J=7.2Hz,1H),6.85-6.80(m,1H),6.72(t,J=6.0Hz,1H),4 .94(dd,J=12.2,5.6Hz,1H),3.96(d,J=5.9Hz,2H),3.81-3.28(m,16H),2.96-2.76(m,3H),2.14(dd,J=10.6,5.5Hz,1H).ESI-MSm / z 532[M+H] + .

[0110] The S-82 can be synthesized using the same method. 1 H NMR(400MHz,Chloroform-d)δ7.93(s,1H),7.58-7.44(m,1H),7.10(d,J=6.7Hz,1H),6.88(d,J=8.4Hz,1H),6.23(s,1H),4.91( dd,J=12.2,5.3Hz,1H),3.43-3.13(m,4H),2.95-2.68(m,3H),2.19-2.09(m,1H),1.78-1.58(m,4H),1.51-1.35(m,4H).ESI-MS m / z456[M+H] + .

[0111]

[0112] Synthesis of compounds S84, S85, S86, and S87. SO2Cl2 (1.5 mL, 21 mmol, 4.2 eq) was slowly added dropwise to S85-1 (0.92 g, 5 mmol, 4.2 eq) at 60 °C. The mixture was heated to 90 °C and reacted for 14 h. The product S85-2 (0.84 g, quantitive) was obtained by direct evaporation and used directly in subsequent reactions. 1 ¹H NMR (400MHz, Acetone-d6) δ 8.18 (dd, J = 8.4, 4.5Hz, 1H), 7.88 (dd, J = 7.2, 2.3Hz, 1H), 7.83 (td, J = 8.7, 2.3Hz, 1H). S85-2 (840mg, 5mmol, 1eq), S80-2 (830mg, 5mmol, 1eq) and AcONa (1.62g, 16mmol, 3.2eq) were dissolved in 7mL AcOH, heated to 140℃ and reacted for 12 minutes. The solvent was then directly evaporated and column chromatography was performed to obtain the key intermediate compound S85-3 (680mg, 49%). 1 H NMR (400MHz, DMSO-d6) δ11.15 (s, 1H), 8.32-7.53 (m, 3H), 5.17 (dd, J = 12.8, 5.4Hz ,1H),2.90(ddd,J=18.6,13.6,5.3Hz,1H),2.70-2.52(m,2H),2.26-2.00(m,1H).

[0113] S85-3 (81 mg, 0.3 mmol, 1 eq), L1 (72 mg, 0.3 mmol, 1 eq), and DIEA (99 μL, 0.6 mmol, 2 eq) were dissolved in 1 mL of DMF, and the mixture was heated to 90 °C and reacted for 12 h until the starting material disappeared. 20 mL of EtOAc was added and the mixture was washed with water. The organic phase was dried, evaporated to dryness, and the product S-85 (45 mg, 32%) was separated by column chromatography. 1 H NMR(400MHz,Chloroform-d)δ7.97(s,1H),7.62(d,J=8.3Hz,1H),6.99(d,J=2.2Hz,1H),6.78(dd,J=8.4,2.2Hz,1H),5.14(t,J =4.8Hz,1H),4.93(dd,J=12.3,5.3Hz,1H),3.82-3.59(m,12H),3.49-3.33(m,4H),2.94-2.69(m,3H),2.17-2.08(m,1H).ESI-MS m / z 475[M+

[0114] H] + .

[0115] S86, S87, and S88 can be prepared using the same method, with S85-3 and different connecting arms L2, L5, and L6 as raw materials.

[0116]

[0117] Synthesis of compounds S88 and S89. S89-1 (1.5 g, 9 mmol, 1 eq), S80-2 (1.5 g, 9 mmol, 1 eq), and TEA (1.39 mL, 10 mmol, 1.1 eq) were added to a flask, and a Dean-Stark apparatus was placed on top. The mixture was refluxed for 12 h. The solvent was evaporated to dryness, and the product S89-2 (1.5 g, 61%) was obtained directly by column chromatography. 1¹H NMR (400MHz, DMSO-d6) δ 11.21 (brs, 1H), 11.10 (s, 1H), 7.65 (t, J = 7.8 Hz, 1H), 7.29 (dd, J = 17.9, 7.8 Hz, 2H), 5.08 (dd, J = 12.9, 5.4 Hz, 1H), 2.99–2.79 (m, 1H), 2.67–2.54 (m, 2H), 2.15–1.89 (m, 1H). S89-2 (83 mg, 0.3 mmol, 1 eq), L3 (100 mg, 0.36 mmol, 1.2 eq), K₂CO₃ (50 mg, 0.6 mmol, 2 eq) and KI (5 mg, 0.03 mmol, 0.1 eq) were dissolved in 2 mL of DMF and heated to 80 °C for 5 h. After filtration, the solvent was evaporated to dryness, and column chromatography was performed to obtain product S-89 (40 mg, 33%). 1 H NMR(400MHz,Chloroform-d)δ8.02(s,1H),7.68(t,J=7.9Hz,1H),7.47(d,J=7.2Hz,1H),7.28(d,J=8.3Hz,1H),4.95(dd,J=12.3,5.4Hz,1H),4.36(t,J=4.9 Hz,2H),3.95(t,J=4.9Hz,2H),3.79(dd,J=5.9,3.4Hz,2H),3.68(dd,J=8.4,3 .5Hz,8H),3.38(t,J=5.1Hz,2H),2.96-2.61(m,3H),2.22-2.06(m,1H).ESI-MS m / z 476[M+H] + .

[0118] The S-88 can also be synthesized using the same method. 1 H NMR(400MHz,Chloroform-d)δ7.98(s,1H),7.68(t,J=7.9Hz,1H),7.46(d,J=7.2Hz,1H),7.21(d,J=8.4Hz,1H),4.96(dd,J=12.2,5.4Hz,1H),4.19 (t,J=6.4Hz,2H),3.30(t,J=6.8Hz,2H),2.96-2.67(m,3H),2.21-2.07(m ,1H),1.90(p,J=6.5Hz,2H),1.73-1.60(m,2H),1.58-1.43(m,4H).ESI-MS m / z 400[M+H] + .

[0119]

[0120] Synthesis of compounds S90 and S91. Using S91-1 as the starting material, intermediate S90-4 was synthesized by referring to the procedures described in Bioorg. Med. Chem. 2011, 19, 4953-4970. 1 ¹H NMR (400MHz, DMSO-d⁶) δ 7.75 (d, J = 7.9 Hz, 1H), 7.16 (d, J = 8.2 Hz, 2H), 5.08 (dd, J = 12.8, 5.4 Hz, 1H), 2.88 (ddd, J = 16.4, 13.4, 5.0 Hz, 1H), 2.69–2.52 (m, 2H), 2.08–1.97 (m, 1H). Intermediates S91-2 (83 mg, 0.3 mmol, 1 eq), L₃ (100 mg, 0.36 mmol, 1.2 eq), K₂CO₃ (50 mg, 0.6 mmol, 2 eq) and KI (5 mg, 0.03 mmol, 0.1 eq) were dissolved in 2 mL of DMF and reacted at 80 °C for 5 h. After filtration, the solvent was evaporated to dryness, and column chromatography was performed to obtain product S-91 (62 mg, 51%). 1 HNMR(400MHz,Chloroform-d)δ8.16(s,1H),7.78(d,J=8.3Hz,1H),7.37(d,J=2.4Hz,1H),7.23(dd,J=8.3,2.4Hz,1H),4.96(dd,J=12.4,5 .3Hz,1H),4.26(t,J=4.7Hz,2H),3.91(t,J=4.7Hz,2H),3.83-3.54(m,10H),3.39(t,J=5.1Hz,2H),2.90-2.68(m,3H),2.21-2.11(m,1H).

[0121] The S-90 was also synthesized using the same method described above. , 1 H NMR(400MHz,Chloroform-d)δ8.03(s,1H),7.78(d,J=8.3Hz,1H),7.34(d,J=2.3Hz,1H),7.19(dd,J=8.4,2.3Hz,1H),4.96(dd,J=12.5,5.4Hz,1 H),4.09(t,J=6.4Hz,2H),3.30(t,J=6.8Hz,2H),2.96-2.65(m,3H),2.2 2-2.11(m,1H),1.90-1.77(m,2H),1.73-1.60(m,2H),1.57-1.41(m,4H).

[0122]

[0123] Synthesis of S92, S93, and S94. The synthesis of compounds S92, S93, and S94 was carried out according to the relevant procedures in Eur. J. Med. Chem. 2018, 151, 237-247.

[0124]

[0125] Synthesis of S95 and S96. S95-1 (86 mg, 0.2 mmol, 1 eq) was dissolved in 2 mL of DMF with 6-azidohexanoic acid (34 mg, 0.2 mmol, 1 eq), and HATU (114 mg, 0.3 mmol, 1.5 eq) and DIEA (71 μL, 0.4 mmol, 2 eq) were added. After reacting for 10 min, water and DCM were added and the mixture was separated. The organic phase was dried over MgSO4 and then evaporated to dryness. The product S-95 (51 mg, 46%) was purified by column chromatography. 1 H NMR(400MHz,Chloroform-d)δ8.80(s,1H),7.37(s,4H),6.08(d,J=9.2Hz,1H),4.73(t,J= 7.8Hz,1H),4.68-4.43(m,3H),4.34(dd,J=14.6,4.5Hz,1H),4.13(d,J=12.3Hz,1H),3.65 -3.56(m,1H),3.49(d,J=1.6Hz,1H),3.26(t,J=6.8Hz,2H),2.69-2.44(m,4H),2.22(t,J= 7.5Hz,2H),2.19-2.10(m,1H),1.72-1.54(m,4H),1.43-1.31(m,2H),0.93(s,9H).ESI-MS m / z 556[M+H] + .

[0126] The S-95 can be synthesized using a similar method. 1H NMR(400MHz,Chloroform-d)δ8.63(s,1H),7.41-7.29(m,2H),7.23(dd,J=15.1,6.5Hz,2H),4.65(t,J=7.9Hz,1H),4.53-4.42(m,3H),4.27(dd,J= 15.0,5.5Hz,1H),4.05-3.86(m,3H),3.67-3.52(m,10H),3.34-3.24(m,2 H),2.50-2.39(m,4H),2.04(dd,J=13.8,7.7Hz,1H),0.89(s,9H).ESI-MS m / z 632[M+H] + .

[0127]

[0128] Synthesis of S97 and S98. Ubenimex (585 mg, 1.9 mmol, 1 eq) was dissolved in H2O / THF solution and added to a THF solution of Fmoc-Cl (578 mg, 2.2 mmol, 1.15 eq) and K2CO3 (591 mg, 4.3 mmol, 2.3 eq). The mixture was heated to room temperature and reacted for 24 h. After acidification with 2 M HCl and solvent removal by evaporation, the residue was extracted with EtOAc. The organic phases were combined, washed with brine, dried, evaporated to dryness, and separated by column chromatography to obtain product S97-1 (530 mg, 52%). ESI-MS m / z 531 [M+H] + S97-1 (100 mg, 0.2 mmol, 1 eq) was further dissolved in 2 mL of DMF with L2 (46 mg, 0.32 mmol, 1 eq), and HATU (184 mg, 0.48 mmol, 1.5 eq) and DIEA (115 μL, 0.64 mmol, 2 eq) were added. After reacting for 10 min, water and DCM were added and the mixture was separated. The organic phase was dried over MgSO4 and then evaporated to dryness. The product S97-2 ​​was purified by column chromatography. ESI-MS m / z 655 [M+H] + Compound S97-2 ​​(100 mg, 0.15 mmol, 1 eq) was dissolved in 4 mL of anhydrous CH2Cl2, and 1.5 mL of Et2N was added and stirred overnight. The solution was then directly evaporated to dryness and subjected to column chromatography to obtain product S-97 (58 mg, 89%). 1H NMR (400MHz, Chloroform-d) δ7.56 (d, J = 8.7Hz, 1H), 7.39-7.31 (m, 2H), 7.30-7.12 (m, 4H), 6. 44(t,J=6.1Hz,1H),4.51-4.35(m,1H),3.99(d,J=3.2Hz,1H),3.56(dt,J=8.8,4.1Hz,1H),3.3 2-3.14(m,4H),3.00(dd,J=13.4,5.1Hz,1H),2.56(dd,J=13.7,9.5Hz,1H),1.80-1.71(m,1H), 1.68-1.55(m,4H),1.54-1.44(m,2H),1.42-1.29(m,4H),0.95(dd,J=12.6,6.2Hz,6H).ESI-MS m / z 433[M+H] + .

[0129] S-98 can be synthesized using a similar method. S-98, 1 H NMR(400MHz,Chloroform-d)δ7.49(d,J=8.6Hz,1H),7.38-7.05(m,6H),6.83(t,J=5.9Hz,1H),4.37(td,J=8.9,4.9Hz,1H),3.94(d,J=3.2Hz,1H),3 .67-3.45(m,12H),3.46-3.22(m,5H),2.93(dd,J=13.6,5.5Hz,1H),2.53 (dd,J=13.7,9.3Hz,1H),1.73-1.48(m,3H),0.86(t,J=7.5Hz,6H).ESI-MS m / z509[M+H] + .

[0130] Example 4 Synthesis of PROTACs-type alkaloid derivatives

[0131] The propynyl key intermediate compounds (compounds 1-4) prepared in Example 1 were reacted with the PROTACs-type azide fragments prepared in Example 3 under the same operating conditions as in Example 2, and then treated to obtain PROTACs-type alkaloid derivatives. The structural characterization of the obtained PROTACs-type alkaloid derivatives is as follows:

[0132]

[0133] 3-(4-((9,10-bis((3,5-dimethylbenzyl)oxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2H-pyrido[2,1-a]isoquinolin-2-yl)methyl)-1H-1,2,3-triazol-1-yl)-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)propa namide(93).38mg,65%yield.Pale yellow crystal,mp.162-164℃. 1 H NMR(400MHz,DMSO-d6)δ11.17(s,1H),9.91(s,1H),8.34(d,J=8.3Hz,1H),8.09-7.86(m,1H),7.81(t,J=7.9Hz,1H),7.63(d,J=7.3Hz,1H),7.22-6.73(m,8H),5.14(dd,J=12.8,5.4Hz,1H),4.98(s,4H),4.65(t,J=6.8Hz,2H),4.12(q,J=5.2Hz,1H),3.17-3.07(m,3H),3.05-2.51(m,7H),2.25(d,J=4.4Hz,12H),2.12-1.98(m,2H),1.91(s,1H),1.73-1.31(m,4H),1.21-1.04(m,1H). 13 CNMR(101MHz,DMSO)δ173.2,170.2,169.7,167.1,137.8,137.8,136.4,132.0,129.6,127.4,125.8,125.6,119.2,118.1,114.9,71.6,70.7,49.4,49.1,36.9,31.4,22.5,21.4,14.5.ESI-MS m / z 894[M+H] + ,HR-EI-MS:m / z 894.4188[M+H] + ,(calcd for C 51 H 56 N7O8 + ,894.4185).

[0134]

[0135] 11-(4-((9,10-bis((3,5-dimethylbenzyl)oxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2H-pyrido[2,1-a]isoquinolin-2-yl)methyl)-1H-1,2,3-triazol-1-yl)-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)unde canamide(94).26mg,50%yield.Orange crystal,mp.113-115℃. 1 H NMR(400MHz,DMSO-d6)δ11.17(s,0.28H),9.68(s,0.07H),8.49(d,J=8.2Hz,1H),7.90-7.75(m,2H),7.60(d,J=7.3Hz,1H),7.04(d,J=6.2Hz,4H),6.93(s,2H),6.80(d,J=12.9Hz,2H),5.15(dd,J=12.8,5.6Hz,1H),4.96(d,J=3.9Hz,4H),4.28(t,J=7.0 Hz,2H),4.04(d,J=16.9 Hz,1H),3.02-2.54(m,8H),2.44(t,J=7.5 Hz,2H),2.25(d,J=3.2 Hz,12H),2.13-1.85(m,3H),1.80-1.73(m,2H),1.64-1.56(m,2H),1.42(s,2H),1.25-1.09(m,12H). 13 C NMR(101MHz,DMSO)δ173.2,173.1,172.5,172.4,170.2,170.1,168.2,167.1,137.7,137.0,136.9,136.6,131.9,129.6,129.5,126.4,125.8,125.6,123.8,118.7,117.2,114.8,113.0,71.1,70.8,49.5,49.4,37.0,31.4,30.2,29.3,29.2,29.2,29.0,28.8,26.3,25.2,22.4,21.4,14.3.ESI-MS m / z 1006[M+H] + ,HR-EI-MS:m / z 1006.5452[M+H] + ,(calcd for C59 H 72 N7O8 + ,1006.5437).

[0136]

[0137] 14-(4-(((9,10-bis((3,5-dimethylbenzyl)oxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2H-pyrido[2,1-a]isoquinolin-2-yl)methyl)-1H-1,2,3-triazol-1-yl)-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-3,6,9,12-tetraoxatetradecanamide(95).28 mg,46%yield.Pale yellow crystal,mp.117-119℃. 1 H NMR(400MHz,DMSO-d6)δ11.17(s,1H),10.34(s,1H),8.71(d,J=8.4 Hz,1H),7.85(t,J=7.9 Hz,2H),7.62(d,J=7.3Hz,1H),7.04(s,4H),7.02-6.70(m,4H),5.16(dd,J=12.8,5.4 Hz,1H),4.98(s,4H),4.46(t,J=5.4 Hz,2H),4.19(s,2H),4.13(q,J=5.3 Hz,1H),3.80-3.69(m,4H),3.69-3.60(m,2H),3.53-3.38(m,8H),2.96-2.52(m,7H),2.25(s,12H),2.15-1.81(m,3H),1.71-1.12(m,5H). 13 C NMR(101 MHz,DMSO)δ173.2,170.2,169.8,168.7,167.1,137.8,137.0,136.4,131.7,129.6,125.8,125.6,124.8,118.8,116.5,71.2,70.7,70.3,70.2,70.1,70.0,69.3,49.6,49.4,40.6,40.4,31.4,22.4,21.4,14.3.ESI-MS m / z1056[M+H] +,HR-EI-MS:m / z1056.5083[M+H] + ,(calcd for C 58 H 71 N7O 12 + ,1056.5077).

[0138]

[0139] 5-((6-(4-((9,10-bis((3,5-dimethylbenzyl)oxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2H-pyrido[2,1-a]isoquinolin-2-yl)methyl)-1H-1,2,3-triazol-1-yl)hexyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione(96).15 mg(from 2,20 mg),43%yield.Light yellow crystal,mp.131-133℃. 1 H NMR(400 MHz,Methanol-d4)δ7.82(s,1H),7.52(d,J=8.4 Hz,1H),7.05(d,J=5.1 Hz,4H),6.93(d,J=14.9 Hz,3H),6.84(d,J=9.3 Hz,2H),6.77(dd,J=8.4,2.3 Hz,1H),5.02(d,J=8.6 Hz,5H),4.61(d,J=7.9 Hz,1H),4.42(t,J=6.9Hz,2H),3.65(d,J=13.3 Hz,1H),3.44(s,1H),3.26(s,1H),3.19-2.91(m,5H),2.90-2.78(m,3H),2.77-2.66(m,2H),2.33-2.18(m,12H),2.18-2.05(m,3H),1.95(p,J=7.0 Hz,2H),1.85(t,J=13.4 Hz,1H),1.76(d,J=14.4 Hz,1H),1.65-1.58(m,2H),1.56(d,J=7.1 Hz,3H),1.49-1.41(m,2H),1.39-1.33(m,2H). 13CNMR(101 MHz,MeOD)δ173.2,170.3,168.2,167.9,154.7,148.7,147.8,137.7,136.9,134.5,129.1,129.0,125.2,125.0,124.8,116.5,115.3,114.5,112.8,105.2,71.5,70.8,68.9,57.4,51.1,49.8,48.9,42.4,40.3,39.1,36.2,30.8,29.7,28.1,26.1,25.9,25.7,22.4,20.0,20.0,13.0.ESI-MS m / z 922[M+H] + ,HR-EI-MS:m / z922.4850[M+H] + ,(calcd for C 54 H 64 N7O7 + ,922.4862).

[0140]

[0141] 5-((2-(2-(2-(2-(4-((9,10-bis((3,5-dimethylbenzyl)oxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2H-pyrido[2,1-a]isoquinolin-2-yl)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione(97).20 mg(from 2,20 mg),52%yield.Green crystal,mp.117-119℃. 1HNMR(400 MHz,Methanol-d4)δ7.86(s,1H),7.52(d,J=8.3 Hz,1H),7.09-6.93(m,6H),6.90-6.75(m,3H),5.11-4.94(m,5H),4.67-4.60(m,1H),4.55(t,J=5.1 Hz,2H),3.87(t,J=5.1 Hz,2H),3.72-3.46(m,11H),3.48-3.40(m,1H),3.28-3.20(m,1H),3.12-3.04(m,1H),3.01-2.93(m,1H),2.92-2.76(m,3H),2.76-2.63(m,2H),2.28(s,12H),2.18-2.00(m,3H),1.85(t,J=13.0 Hz,1H),1.76(d,J=14.3 Hz,1H),1.56(d,J=7.1 Hz,3H). 13 C NMR(101 MHz,MeOD)δ173.2,170.3,168.2,167.8,154.7,147.8,137.7,137.7,136.9,134.4,129.0,129.0,125.2,125.0,124.8,117.0,115.4,114.4,112.7,105.7,71.4,70.8,70.1,70.1,70.0,69.1,68.9,57.3,51.0,49.9,48.9,42.6,40.2,39.2,36.6,30.8,26.1,22.4,20.0,20.0,13.4.ESI-MS m / z 998[M+H] + ,HR-EI-MS:m / z 998.5032[M+H] + ,(calcd for C 56 H 68 N7O 10 + ,998.5022).

[0142]

[0143] N-(6-(4-((9,10-bis((3,5-dimethylbenzyl)oxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2H-pyrido[2,1-a]isoquinolin-2-yl)methyl)-1H-1,2,3-triazol-1-yl)hexyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)acetamide(98).10 mg(from 2,12 mg),41%yield.Green crystal,mp.218-220℃. 1 H NMR(400MHz,Methanol-d4)δ7.82(s,1H),7.57(dd,J=8.3,3.4 Hz,1H),7.04(d,J=12.3 Hz,4H),6.93(dd,J=11.2,3.4Hz,3H),6.87(d,J=8.4 Hz,3H),5.10-4.96(m,5H),4.76(d,J=10.4 Hz,1H),4.41-4.26(m,2H),3.87(s,2H),3.74(s,1H),3.64-3.52(m,1H),3.45-3.37(m,1H),3.28-2.97(m,5H),2.92(s,2H),2.86-2.76(m,1H),2.75-2.58(m,2H),2.28(d,J=4.3 Hz,13H),2.15(dd,J=14.7,6.4 Hz,1H),2.07-2.02(m,1H),2.00-1.89(m,1H),1.87-1.75(m,3H),1.62(d,J=7.3 Hz,3H),1.47-1.41(m,2H),1.27-1.19(m,4H). 13C NMR(101MHz,MeOD)δ173.2,170.8,170.3,167.9,167.7,153.9,137.7,137.7,136.9,134.3,129.0,129.0,125.2,125.0,124.7,118.6,116.5,114.4,112.7,105.6,71.4,70.8,57.9,51.5,49.7,49.0,46.3,39.7,39.0,38.4,35.9,30.8,29.7,28.6,25.5,22.4,20.0,20.0,13.0.ESI-MS m / z 1055[M+H] + ,HR-EI-MS:m / z 1055.5251[M+H] + ,(calcd forC 58 H 71 N8O 11 + ,1055.5237).

[0144]

[0145] N-(2-(2-(2-(2-(4-((9,10-bis((3,5-dimethylbenzyl)oxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2H-pyrido[2,1-a]isoquinolin-2-yl)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)acetamide(99).11 mg(from 2,12 mg),49%yield.Green crystal,mp.158-160℃. 1H NMR(400 MHz,Methanol-d4)δ7.88(s,1H),7.59(d,J=8.3 Hz,1H),7.05(d,J=7.0 Hz,4H),6.96(d,J=2.1 Hz,3H),6.93-6.78(m,3H),5.04(t,J=11.2 Hz,5H),4.73(d,J=10.8 Hz,1H),4.56(t,J=5.1Hz,2H),3.99-3.79(m,4H),3.72(s,1H),3.65-3.41(m,11H),3.41-3.36(m,3H),3.15-2.99(m,2H),2.91(s,2H),2.84-2.76(m,1H),2.74-2.60(m,2H),2.29(s,13H),2.17-2.10(m,1H),2.09-2.02(m,1H),1.98-1.88(m,1H),1.80(d,J=15.0 Hz,1H),1.61(d,J=7.0 Hz,3H). 13 C NMR(101 MHz,MeOD)δ173.2,170.8,170.2,167.9,167.7,154.0,148.9,147.9,137.7,137.7,136.9,134.3,129.1,129.0,125.2,125.0,124.8,118.6,116.3,114.4,105.9,71.3,70.8,70.1,70.0,69.9,69.9,69.1,69.0,68.7,58.1,51.3,49.9,49.0,48.3,46.3,40.0,39.0,38.9,35.1,30.8,25.8,22.4,20.0,20.0,13.1.ESI-MS m / z 979[M+H] + ,HR-EI-MS:m / z 979.5085[M+H] + ,(calcd for C 56 H 67 N8O8 + ,979.5076).

[0146]

[0147] 4-((6-(4-((9,10-bis((3,5-dimethylbenzyl)oxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2H-pyrido[2,1-a]isoquinolin-2-yl)methyl)-1H-1,2,3-triazol-1-yl)hexyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione(100).36 mg(from 2,34 mg),60%yield.Orange crystal,mp.121-123℃. 1 H NMR(400MHz,Methanol-d4)δ7.81(d,J=2.3 Hz,1H),7.51(t,J=7.8 Hz,1H),7.10-6.93(m,8H),6.90-6.76(m,2H),5.09-4.96(m,5H),4.58(d,J=11.2 Hz,1H),4.40(t,J=6.9 Hz,2H),3.60(t,J=6.8 Hz,1H),3.45-3.37(m,1H),3.30-3.14(m,3H),3.12-3.00(m,1H),3.00-2.90(m,1H),2.88(s,2H),2.83-2.73(m,1H),2.74-2.60(m,2H),2.28(d,J=3.1 Hz,12H),2.18-2.01(m,3H),1.93(p,J=6.6 Hz,2H),1.83(t,J=13.4 Hz,1H),1.75(d,J=14.3 Hz,1H),1.64-1.57(m,2H),1.54(d,J=7.1 Hz,3H),1.49-1.40(m,2H),1.38-1.32(m,2H). 13CNMR(101 MHz,MeOD)δ173.2,170.2,169.4,167.8,148.6,147.8,146.8,137.7,136.9,135.8,132.5,129.0,129.0,125.2,125.0,123.9,116.6,114.5,112.7,110.4,109.6,71.4,70.8,69.0,57.2,51.0,49.7,48.8,41.7,40.3,39.2,36.6,30.8,29.7,28.5,26.3,25.8,25.7,22.4,20.0,20.0,14.4.ESI-MS m / z 922[M+H] + ,HR-EI-MS:m / z 922.4870[M+H] + ,(calcd for C 54 H 64 N7O7 + ,922.4862).

[0148]

[0149] 4-((2-(2-(2-(2-(4-((9,10-bis((3,5-dimethylbenzyl)oxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2H-pyrido[2,1-a]isoquinolin-2-yl)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione(101).21 mg(from 2,20 mg),55%yield.Yellow crystal,mp.122-123℃. 1HNMR(400 MHz,Methanol-d4)δ7.92(s,1H),7.51(t,J=7.8 Hz,1H),7.19-6.76(m,10H),5.14-4.98(m,5H),4.80(d,J=11.2 Hz,1H),4.54(t,J=5.0 Hz,2H),3.87(t,J=4.8 Hz,2H),3.82-3.74(m,1H),3.74-3.49(m,11H),3.49-3.39(m,3H),3.17-3.01(m,2H),2.91(s,2H),2.84-2.76(m,1H),2.75-2.62(m,2H),2.28(d,J=2.6 Hz,12H),2.20-2.00(m,3H),1.98-1.78(m,2H),1.64(d,J=7.5 Hz,3H). 13 C NMR(101 MHz,MeOD)δ173.2,170.7,169.3,167.8,149.0,148.1,146.7,137.7,137.7,136.8,135.9,132.4,129.1,129.0,125.2,124.9,116.8,114.3,112.5,110.7,109.8,70.7,70.2,70.2,70.1,70.0,69.1,56.3,50.3,48.8,48.4,41.8,39.8,38.8,36.4,30.9,25.5,22.3,20.0,20.0,14.6.ESI-MS m / z 998[M+H] + ,HR-EI-MS:m / z 998.5028[M+H] + ,(calcd forC 56 H 68 N7O 10 + ,998.5022).

[0150]

[0151] N-(6-(4-((9,10-bis((3,5-dimethylbenzyl)oxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2H-pyrido[2,1-a]isoquinolin-2-yl)methyl)-1H-1,2,3-triazol-1-yl)hexyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)acetamide(102).39 mg(from 2,34 mg),61%yield.Yellow crystal,mp.143-145℃. 1 HNMR(400MHz,Methanol-d4)δ7.80(d,J=2.9 Hz,1H),7.54(t,J=7.8 Hz,1H),7.14-7.01(m,5H),6.95(s,2H),6.90-6.80(m,3H),5.10-4.97(m,5H),4.64(d,J=8.7 Hz,1H),4.34(t,J=8.1 Hz,2H),3.92(s,1H),3.69-3.61(m,1H),3.53-3.38(m,1H),3.31-3.22(m,1H),3.17(p,J=6.8 Hz,2H),3.12-3.04(m,1H),3.03-2.96(m,1H),2.90(s,2H),2.86-2.77(m,1H),2.76-2.61(m,2H),2.28(d,J=2.7 Hz,13H),2.15-2.03(m,2H),1.93-1.73(m,4H),1.57(d,J=7.0 Hz,3H),1.48-1.39(m,2H),1.29-1.22(m,4H). 13C NMR(101 MHz,MeOD)δ173.2,170.4,170.2,169.0,167.7,148.7,147.8,145.8,137.7,136.9,135.9,132.5,129.0,129.0,125.2,125.0,123.9,116.7,114.4,112.7,111.4,111.0,100.0,71.4,70.8,69.0,57.4,51.1,49.6,48.8,47.0,45.5,40.3,39.2,38.4,36.6,30.8,29.6,28.6,26.2,25.4,25.3,22.4,20.0,20.0,14.4.ESI-MS m / z 922[M+H] + ,HR-EI-MS:m / z 979.5068[M+H] + ,(calcd for C 56 H 67 N8O8 + ,979.5076).

[0152]

[0153] N-(2-(2-(2-(2-(4-((9,10-bis((3,5-dimethylbenzyl)oxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2H-pyrido[2,1-a]isoquinolin-2-yl)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)acetamide(103).18 mg(from 2,20 mg),45%yield.Light yellowcrystal,mp.119-121℃. 1H NMR(400 MHz,Methanol-d4)δ7.87(s,1H),7.54(t,J=7.8 Hz,1H),7.15-6.98(m,5H),6.94(s,2H),6.85(t,J=6.5 Hz,3H),5.14-4.95(m,5H),4.64(d,J=12.8 Hz,1H),4.55(t,J=5.1 Hz,2H),3.93(s,2H),3.87(t,J=5.1 Hz,2H),3.68-3.60(m,1H),3.59-3.36(m,13H),3.30-3.20(m,1H),3.13-3.03(m,1H),2.97(d,J=16.1 Hz,1H),2.89(s,2H),2.84-2.75(m,1H),2.75-2.62(m,2H),2.28(d,J=1.6 Hz,12H),2.23-2.17(m,1H),2.15-2.03(m,2H),1.88(t,J=12.9 Hz,1H),1.76(d,J=15.4 Hz,1H),1.56(d,J=7.1 Hz,3H). 13 C NMR(101 MHz,MeOD)δ173.2,170.4,170.1,169.0,167.7,148.6,147.8,145.9,137.7,137.7,136.9,136.9,135.9,132.4,129.1,129.0,125.2,125.0,116.8,114.4,112.7,111.4,110.9,71.4,70.8,70.1,70.0,70.0,69.8,69.1,69.0,57.4,51.1,49.9,48.8,45.4,40.3,39.2,39.0,36.4,30.8,26.2,22.4,20.0,20.0,14.4.ESI-MS m / z 1055[M+H] + ,HR-EI-MS:m / z 1055.5249[M+H] + ,(calcd forC 58 H 71 N8O 11 + ,1055.5237)..

[0154]

[0155] 4-((6-(4-((9,10-bis((3,5-dimethylbenzyl)oxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2H-pyrido[2,1-a]isoquinolin-2-yl)methyl)-1H-1,2,3-triazol-1-yl)hexyl)oxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione(104).40 mg(from 2,35 mg),65%yield.Green crystal,mp.127-129℃. 1 H NMR(400MHz,Methanol-d4)δ7.87(s,1H),7.75-7.67(m,1H),7.40(t,J=7.4 Hz,1H),7.33(dd,J=8.6,3.7 Hz,1H),7.04(d,J=10.8 Hz,4H),6.94(d,J=9.8 Hz,2H),6.84(dd,J=9.0,4.2Hz,2H),5.12-4.97(m,5H),4.66(d,J=15.8 Hz,1H),4.42(t,J=7.0 Hz,2H),4.11(q,J=6.3 Hz,2H),3.72-3.63(m,1H),3.56-3.45(m,1H),3.32-3.26(m,1H),3.15-2.96(m,2H),2.90(s,2H),2.85-2.74(m,1H),2.73-2.58(m,2H),2.27(d,J=6.5 Hz,12H),2.18-2.02(m,3H),2.02-1.86(m,3H),1.84-1.70(m,3H),1.59(d,J=7.1 Hz,3H),1.58-1.48(m,2H),1.43-1.35(m,2H). 13C NMR(101 MHz,MeOD)δ173.1,170.2,167.2,166.1,156.5,148.9,148.0,137.7,137.7,136.9,136.6,133.6,129.0,125.1,125.0,124.9,119.0,114.9,114.3,71.2,70.7,68.8,68.6,58.1,51.5,49.7,49.0,39.9,38.9,36.4,30.7,29.6,28.2,25.7,25.5,25.0,22.3,20.0,20.0,14.7.ESI-MS m / z 923[M+H] + ,HR-EI-MS:m / z 823.4710[M+H] + ,(calcd for C 54 H 63 N6O8 + ,923.4702).

[0156]

[0157] 5-((6-(4-((9,10-bis((3,5-dimethylbenzyl)oxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2H-pyrido[2,1-a]isoquinolin-2-yl)methyl)-1H-1,2,3-triazol-1-yl)hexyl)oxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione(105).37 mg(from 2,34 mg),62%yield.Pale yellow crystal,mp.126-127℃. 1H NMR(400 MHz,Methanol-d4)δ7.85(s,1H),7.74(d,J=8.3 Hz,1H),7.31(s,1H),7.23(dd,J=8.3,2.3 Hz,1H),7.05(s,4H),6.95(s,2H),6.84(d,J=11.7 Hz,2H),5.13-5.07(m,1H),5.02(d,J=6.1 Hz,6H),4.65(d,J=11.5 Hz,1H),4.44(t,J=7.2 Hz,2H),4.05(t,J=6.3Hz,2H),3.76-3.61(m,1H),3.57-3.43(m,1H),3.31-3.24(m,1H),3.17-2.96(m,2H),2.95-2.79(m,3H),2.79-2.63(m,2H),2.28(s,12H),2.17-2.06(m,3H),2.02-1.93(m,2H),1.86(d,J=12.6 Hz,1H),1.82-1.71(m,3H),1.58(d,J=7.2 Hz,3H),1.56-1.46(m,2H),1.43-1.35(m,2H). 13 C NMR(101 MHz,MeOD)δ173.1,170.1,167.2,167.2,164.5,148.8,147.8,137.7,136.9,134.2,131.9,129.1,129.0,125.1,124.9,124.9,123.2,120.0,114.4,112.7,108.6,71.4,70.8,68.8,68.5,57.6,51.2,49.8,49.2,40.2,39.1,36.4,30.8,29.6,28.4,26.0,25.7,25.0,22.3,20.0,20.0,14.7.ESI-MS m / z 923[M+H] + ,HR-EI-MS:m / z923.4703[M+H] + ,(calcd for C 54 H 63 N6O8 + ,923.4702).

[0158]

[0159] 4-(2-(2-(2-(2-(4-((9,10-bis((3,5-dimethylbenzyl)oxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2H-pyrido[2,1-a]isoquinolin-2-yl)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione(106).21 mg(from 2,20 mg),55%yield.Orange crystal,mp.116-118℃. 1 H NMR(400 MHz,Methanol-d4)δ7.89(s,1H),7.71(t,J=7.6 Hz,1H),7.39(dd,J=12.5,7.9 Hz,2H),7.05(d,J=8.1Hz,4H),6.94(s,2H),6.85(d,J=6.4 Hz,2H),5.10-4.96(m,5H),4.59(d,J=11.6 Hz,1H),4.52(t,J=5.0 Hz,2H),4.30(dt,J=5.6,3.1Hz,2H),3.93-3.81(m,4H),3.71(t,J=4.7 Hz,2H),3.62-3.49(m,7H),3.43-3.36(m,1H),3.25-3.15(m,1H),3.10-3.01(m,1H),2.99-2.91(m,1H),2.82(d,J=28.6 Hz,3H),2.75-2.60(m,2H),2.28(s,12H),2.19-2.03(m,3H),1.83(t,J=13.3 Hz,1H),1.74(d,J=15.7Hz,1H),1.55(d,J=7.1 Hz,3H). 13C NMR(101 MHz,MeOD)δ173.1,170.0,167.1,165.9,156.3,148.6,147.8,137.7,137.7,137.0,136.5,133.6,129.0,129.0,125.2,125.0,119.3,116.8,115.3,114.4,112.8,71.3,70.8,70.6,70.1,70.0,69.0,69.0,57.3,50.9,49.9,49.0,40.2,39.3,36.4,30.8,26.3,22.2,20.0,20.0,14.5.ESI-MS m / z 999[M+H] + ,HR-EI-MS:m / z999.4872[M+H] + ,(calcd for C 56 H 67 N6O 11 + ,999.4862).

[0160]

[0161] 5-(2-(2-(2-(2-(4-((9,10-bis((3,5-dimethylbenzyl)oxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2H-pyrido[2,1-a]isoquinolin-2-yl)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione(107).29 mg,51%yield.Orange crystal,mp.105-107℃. 1H NMR(400 MHz,Methanol-d4)δ7.87(s,1H),7.72(d,J=8.3 Hz,1H),7.35(d,J=2.3 Hz,1H),7.24(dd,J=8.3,2.4 Hz,1H),7.04(d,J=5.9 Hz,4H),6.94(s,2H),6.83(d,J=2.7 Hz,2H),5.16-4.97(m,5H),4.64-4.43(m,3H),4.22(t,J=4.5 Hz,2H),3.84(dt,J=13.0,4.8Hz,4H),3.71-3.48(m,9H),3.41-3.35(m,1H),3.24-3.10(m,1H),3.09-2.99(m,1H),2.99-2.84(m,3H),2.84-2.76(m,1H),2.76-2.61(m,2H),2.27(s,12H),2.18-2.02(m,3H),1.82(t,J=13.1 Hz,1H),1.73(d,J=14.8 Hz,1H),1.52(d,J=7.2 Hz,3H). 13 C NMR(101 MHz,MeOD)δ173.1,170.0,167.2,167.1,164.3,148.5,147.7,137.7,137.7,137.0,137.0,134.2,129.0,129.0,125.2,125.0,124.9,124.8,123.5,120.2,114.4,112.8,108.8,71.3,70.8,70.4,70.1,70.0,69.2,69.1,69.0,68.3,57.0,50.8,49.9,49.2,40.4,39.3,36.7,30.8,26.4,22.3,20.1,20.0,14.5.ESI-MS m / z 999[M+H] + ,HR-EI-MS:m / z 999.4871[M+H] + ,(calcd for C 56 H 67 N6O 11 + ,999.4862).

[0162]

[0163] (R)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutanamido)-N-(6-(4-((Rac-(2S,4S,11bR)-9,10-bis((3,5-dimethylbenzyl)oxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2H-pyrido[2,1-a]isoquinolin-2-yl)methyl)-1H-1,2,3-triazol-1-yl)hexyl)-4-methylpentanamide(108).32 mg,58%yield.Yellow crystal,mp.83-85℃. 1 HNMR(400 MHz,Methanol-d4)δ7.75(s,1H),7.36-7.16(m,5H),7.04(s,4H),6.93(s,2H),6.83(s,1H),6.76(s,1H),4.98(s,4H),4.39(dd,J=9.0,5.4 Hz,1H),4.33(t,J=7.0 Hz,2H),4.14(d,J=11.1 Hz,1H),3.95(d,J=3.7 Hz,1H),3.24-3.10(m,3H),2.91(dd,J=13.3,6.9 Hz,3H),2.88-2.62(m,5H),2.27(s,12H),2.06-1.81(m,4H),1.74-1.55(m,5H),1.51-1.42(m,2H),1.33(d,J=8.4 Hz,8H),0.95(dd,J=9.2,5.7 Hz,6H). 13 C NMR(101MHz,MeOD)δ174.0,173.0,147.7,147.1,143.0,138.6,137.6,137.3,137.2,130.8,129.0,129.0,128.9,128.2,127.5,126.1,125.3,125.2,125.0,123.7,114.9,113.3,72.3,71.6,71.0,70.4,55.2,54.9,51.7,49.7,49.6,41.4,40.8,40.1,39.4,38.7,38.4,29.7,28.6,28.1,25.7,25.6,24.7,22.0,20.8,20.1,20.0,13.7.ESI-MS m / z 956[M+H] +,HR-EI-MS:m / z 956.6030[M+H] + ,(calcd for C 57 H 78 N7O6 + ,956.6008).

[0164]

[0165] (R)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutanamido)-N-(2-(2-(2-(2-(4-(((2S,4S,11bR)-9,10-bis((3,5-dimethylbenzyl)oxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2H-pyrido[2,1-a]isoquinolin-2-yl)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)-4-methylpentanamide(109).26 mg,42%yield.Yellowcrystal,mp.65-67℃. 1 H NMR(400 MHz,Methanol-d4)δ7.82(s,1H),7.37-7.17(m,5H),7.05(s,4H),6.95(s,2H),6.83(s,1H),6.77(s,1H),4.99(d,J=3.7 Hz,4H),4.55(d,J=5.1 Hz,2H),4.44(dd,J=8.7,5.3 Hz,1H),4.13(d,J=8.3 Hz,1H),3.94(s,1H),3.87(t,J=5.0 Hz,2H),3.61-3.45(m,10H),3.35-3.33(m,2H),3.18(t,J=6.6 Hz,1H),3.00-2.62(m,8H),2.28(s,12H),2.06-1.89(m,2H),1.62(dt,J=28.0,11.4 Hz,5H),1.33(d,J=7.0 Hz,3H),0.95(t,J=7.0 Hz,6H). 13C NMR(101 MHz,MeOD)δ174.1,173.2,147.7,147.1,143.0,138.8,137.6,137.3,137.3,131.0,129.0,128.9,128.9,128.2,127.6,126.1,125.2,125.0,124.5,114.9,113.4,72.4,71.6,71.1,70.5,70.1,70.1,70.0,69.8,69.0,55.3,54.8,51.6,49.9,49.5,41.3,40.8,40.1,39.5,39.0,38.4,28.2,24.6,22.0,20.8,20.1,20.0,13.7.ESI-MS m / z 1032[M+H] + ,HR-EI-MS:m / z 1032.6122[M+H] + ,(calcd forC 59 H 82 N7O9 + ,1032.6169).

[0166]

[0167] (2S,4R)-1-((S)-2-(6-(4-((Rac-(2S,4S,11bR)-9,10-bis((3,5-dimethylbenzyl)oxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2H-pyrido[2,1-a]isoquinolin-2-yl)methyl)-1H-1,2,3-triazol-1-yl)hexanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide(110).42 mg,67%yield.Pink crystal,125-127℃. 1H NMR(400 MHz,Methanol-d4)δ8.87(s,1H),7.82(s,1H),7.53-7.36(m,4H),7.05(s,4H),6.94(s,2H),6.84(d,J=9.7 Hz,2H),5.01(s,4H),4.68-4.46(m,5H),4.44-4.30(m,3H),3.91(d,J=11.2Hz,1H),3.85-3.73(m,1H),3.54(s,1H),3.20-3.00(m,2H),2.98-2.81(m,3H),2.47(s,3H),2.33-2.04(m,18H),1.96-1.79(m,3H),1.75-1.59(m,3H),1.50(d,J=7.1 Hz,3H),1.37-1.30(m,2H),1.04(s,9H). 13 C NMR(101 MHz,MeOD)δ174.2,173.0,170.9,151.4,148.5,147.7,147.6,142.5,138.9,137.7,137.0,137.0,132.0,130.1,129.0,129.0,128.9,128.0,127.6,125.5,125.2,125.0,123.9,114.6,112.9,71.4,70.9,69.7,69.3,59.4,57.6,56.8,56.6,50.8,49.7,42.3,40.7,39.4,37.6,36.9,35.1,34.9,29.5,26.6,25.7,25.6,24.9,20.1,20.0,14.5,14.2.ESI-MS m / z 1093[M+H] + ,HR-EI-MS:m / z 1093.5969[M+H] + ,(calcd forC 63 H 81 N8O7S + ,1093.5943).

[0168]

[0169] (2S,4R)-1-((S)-14-(4-((Rac-(2S,4S,11bR)-9,10-bis((3,5-dimethylbenzyl)oxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2H-pyrido[2,1-a]isoquinolin-2-yl)methyl)-1H-1,2,3-triazol-1-yl)-2-(tert-butyl)-4-oxo-6,9,12-trioxa-3-azatetradecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide(111).35mg,52%yield.Yellow crystal,mp.100-102℃. 1 H NMR(400MHz,Methanol-d4)δ8.87(s,1H),7.95-7.81(m,1H),7.42(q,J=8.1Hz,4H),7.05(s,4H),6.94(s,2H),6.84(d,J=10.8Hz,2H),5.01(d,J=5.3Hz,4H),4.71(d,J=4.8Hz,1H),4.62-4.41(m,6H),4.35(d,J=15.7Hz,1H),4.03(s,2H),3.96-3.77(m,4H),3.74-3.50(m,8H),3.52-3.42(m,1H),3.31-3.20(m,1H),3.12-2.97(m,2H),2.95-2.78(m,3H),2.52-2.42(m,3H),2.28(s,13H),2.17-2.00(m,3H),1.85-1.63(m,2H),1.47(d,J=7.0Hz,3H),1.05(s,9H). 13CNMR(101MHz,MeOD)δ172.9,170.6,170.2,151.4,148.3,147.6,138.8,137.7,137. 1,137.1,130.1,129.1,129.0,129.0,128.1,127.6,125.9,125.2,125.0,124.8,11 4.7,113.0,71.5,70.9,70.8,70.2,70.0,69.7,69.5,69.1,59.4,56.7,56.4,50.5, 49.9,42.3,40.7,39.5,37.6,37.1,35.7,26.9,25.6,20.1,20.0,14.5,14.2.ESI-MS m / z 1155[M+H] + HR-EI-MS: m / z 1169.6112 [M+H] + ,(calcd for C 69 H 85 N8O 10 S + ,1169.6104).

[0170] Example 5: Identification of molecular targets using biotinylated small molecule probes

[0171] The classic method of target separation, which utilizes the affinity binding of compounds to target proteins, involves immobilizing the active compound on a solid matrix surface to "fish out" the bound protein. Based on the high affinity and low specificity of the biotin-streptavidin reaction system (Adv. Protein Chem. 1975, 29, 85-133, J. Biol. Chem. 2002, 277, 30892-30900), the biotinylated benzoquinolactone alkaloid probe obtained in Example 2 was reacted with the target protein. Mass spectrometry analysis of the resulting specific protein bands revealed that the potential target of this type of compound is the Rab GDI2 protein. Furthermore, RNA interference technology confirmed that this target is an important molecular target inducing paraapoptosis and cellular vacuolation.

[0172] Cells were seeded in 96-well plates at a density of approximately 40–50%. Treatment groups were treated with different concentrations of the compound (0.5, 1, 2, 4 μM) prepared in fresh culture medium, while the control group was treated with an equal volume of DMSO. Incubation was performed for 48 hours, with at least three replicates per group. 10 μL of MTT was added to each well to a final concentration of 0.5 mg / mL. After incubation at 37°C for 4 hours, the supernatant was aspirated and 100 μL of DMSO was added. The mixture was vortexed to mix. The absorbance of each well was measured at 570 nm, and the average of the three replicates was calculated. Cell viability was calculated using the following formula:

[0173]

[0174] The results showed that the IC50 of the biotinylated probe 5 on the inhibitory effect on prostate tumor cells was [missing information]. 50 The concentration was 1.39 μM, and the IC50 of its precursor, benzoquinolidine, was [missing value]. 50 =0.57 μM, a slight decrease compared to the previous value. Simultaneously, this biotin probe also induced strong cytoplasmic vacuolation in cells, indicating that the compound can induce cell death in PC-3 cells through paraapoptosis. Therefore, this probe can maintain the original compound's antitumor activity and corresponding morphological characteristics, and can serve as an effective target probe for subsequent related research.

[0175] Next, this embodiment uses ABPP-SLIAC technology to identify protein targets in the proteome that directly interact with biotinylated probe 5. First, PC-3 cells were cultured for more than 10 passages in "light" and "heavy" SILAC media. A mixture of proteins lysed from "light" and "heavy" PC-3 cells was incubated overnight (4°C) with DMSO or 10 μM biotinylated probe 5. Then, equal volumes of the two groups of light and heavy protein samples were mixed, and target proteins were enriched using streptavidin agarose beads, followed by trypsin digestion. The digested peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS) to identify proteins captured by biotinylated probe 5. The results showed that GDI2 protein may be a potential target of compound benzoquinolone alkaloid 22b.

[0176]

[0177] Next, we continued to investigate the role of these 22b-induced vacuoles. We silenced the GDI2 protein using RNA interference (RNAii) technology, and designed and synthesized GDI2-specific interfering siRNA sequences: Sense: 5'-GGAACCAAUUGAACAGAAAdTdT-3', as shown in SEQ ID NO.1; Antisense: 5'-UUUCUGUUCAAUUGGUUCCdTdT-3', as shown in SEQ ID NO.2. Cells were seeded into 6-well plates and cultured overnight. Optimal interference was achieved at a cell density of 30-50%. An equal amount of NC interfering sequence was added to the control group. The interference was then performed according to the lipofectamine 2000 instructions: the interfering RNA and liposomes were pre-mixed with Opti-MEM and incubated at 37°C for 5 min, then the two were mixed and incubated at 37°C for 15 min. The medium in the 6-well plates was aspirated and replaced with serum-free, antibiotic-free Opti-MEM medium. 200 μL of the mixture of liposomes and interfering RNA was added to the corresponding wells of the 6-well plates, mixed well, and incubated in a cell culture incubator. After 4 hours, the medium was replaced with fresh medium. Subsequent experiments were conducted 48 hours after interference. It was observed that silencing GDI2 induced intracellular vacuolation in PC-3 cells, further validating that GDI2 protein is a key endogenous target for compound 22b. Simultaneously, interfering with GDI2 protein expression also inhibited cell proliferation, producing a similar inhibitory effect to compound 22b in PC-3 cells. Furthermore, the combination of GDI2-siRNA and compound 22b also produced a significant synergistic effect in inhibiting cell proliferation activity, such as… Figure 1 As shown above, these results fully demonstrate that GDI2 is an important molecular target for initiation-related apoptosis and the resulting cytoplasmic vacuolization.

[0178] Example 6: PROTACs-like molecular protein degradation experiment

[0179] Aspc-1 pancreatic tumor cells were treated with PROTACs, and proteins were extracted and Western blotting was performed as described in Example 5. The expression level of GDI2 was analyzed. The results showed that compound 109 significantly induced the degradation of GDI2 in Aspc-1 cells. Figure 2 As shown.

[0180] Example 7: Inhibitory effect of the compound on tumor cells

[0181] As shown in Example 5, the IC50 of all compounds on the inhibitory effect of AsPc-1 in pancreatic tumors was determined. 50 The screening results for the inhibitory effects of preferred compounds on these tumor cells are as follows:

[0182] Table 3. Inhibitory effects of benzo[a]quinoloxetine compounds on AsPc-1 cells.

[0183]

[0184]

[0185] IC in the table 50 Data are from MTT measurements (n=3).

[0186] Example 8: Tumor-inhibiting experiment of alkaloids on tumor-bearing nude mice:

[0187] BALB / c-nu nude mice, 6 weeks old, male, were purchased from the Animal Center of the Chinese Academy of Medical Sciences (Beijing). The experimental protocol for this embodiment was reviewed and approved by the Experimental Animal Nursing Committee of Shandong University. Logarithmically growing AsPc-1 cells were harvested, digested with trypsin, and dispersed into single cells, with the cell concentration adjusted to 1×10⁻⁶. 7 Tumor fluid was injected subaxillarily into nude mice at a rate of 0.1 mL per mouse. The injection was continued until the tumor reached 100 mm in size. 3 Animals were then randomly assigned to four groups: a negative control group, a compound 19 group ((2S,4S,11bR)-9,10-bis(3,5-dimethylbenzyloxy)-2-((1-(4-fluorobenzyl)-1-hydro-1,2,3-triazol-4-yl)methyl)-4-methyl-1,3,4,6,7,11b-hexahydro-2-hydro-pyridano[2,1-a]isoquinoline-2-ol) group (25 mg / kg and 40 mg / kg), a compound 109 group (15 mg / kg), and a positive control group (gemcitabine, 40 mg / kg). All animals received an intraperitoneal injection of 0.1 mL per intraperitoneal injection, once every three days for 18 consecutive days. Tumor size was measured using calipers, and tumor volume (mm) was calculated using the following formula. 3 ):volume=length×(width) 2 ×0.5. At the end of the experiment, the animals were anesthetized, tumor tissue was collected, and tumor weight was measured. The results are as follows: Figure 3 As shown, compounds 19 and 109 were found to significantly inhibit tumor growth, with compound 109 requiring the lowest dosage, indicating that compound 109 has a better anti-pancreatic tumor effect.

[0188] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A benzoquinolone alkaloid compound, characterized in that, Selected from the following compounds: Rac- (2 S 4 S ,11 bR )-3-(4-((9,10-bis(3,5-dimethylbenzyloxy)-2-hydroxy-4-methyl-1,3,4,6,7,11 b -hexahydro-2-hydro-pyridino[2,1-a]isoquinoline-2-yl)methyl)-1-hydro-1,2,3-triazol-1-yl)- N -(2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxoisoindoline-4-yl)propionamide; Rac- (2 S 4 S ,11 bR )-11-(4-((9,10-bis(3,5-dimethylbenzyloxy)-2-hydroxy-4-methyl-1,3,4,6,7,11 b -hexahydro-2-hydro-pyridino[2,1-a]isoquinoline-2-yl)methyl)-1-hydro-1,2,3-triazol-1-yl)- N -(2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxoisoindoline-4-yl)undecanoamide; Rac- (2 S 4 S ,11 bR )-14-(4-(((9,10-bis(3,5-dimethylbenzyloxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2-hydro-pyrido[2,1-a]isoquinoline-2-yl)methyl)-1-hydro-1,2,3-triazol-1-yl)- N -(2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxoisoindoline-4-yl)-3,6,9,12-tetraoxotetradecanoamide; Rac- (2 S 4 S ,11 bR )-5-((6-(4-((9,10-bis(3,5-dimethylbenzyloxy)-2-hydroxy-4-methyl-1,3,4,6,7,11 b -hexahydro-2-hydropyrido[2,1-a]isoquinoline-2-yl)methyl)-1-hydro-1,2,3-triazol-1-yl)n-hexyl)amino)-2-(2,6-dioxo-piperidin-3-yl)isoindole-1,3-dione; Rac- (2 S 4 S ,11 bR )-5-((2-(2-(2-(4-((9,10-bis(3,5-dimethylbenzyloxy)-2-hydroxy-4-methyl-1,3,4,6,7,11 b -hexahydro-2-hydro-pyrido[2,1-a]isoquinoline-2-yl)methyl)-1-hydro-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxo-piperidin-3-yl)isoindole-1,3-dione; Rac- (2 S 4 S ,11 bR )-4-((6-(4-((9,10-bis(3,5-dimethylbenzyloxy)-2-hydroxy-4-methyl-1,3,4,6,7,11 b -hexahydro-2-hydropyrido[2,1-a]isoquinoline-2-yl)methyl)-1-hydro-1,2,3-triazol-1-yl)n-hexyl)amino)-2-(2,6-dioxo-piperidin-3-yl)isoindole-1,3-dione; Rac- (2 S 4 S ,11 bR )-4-((2-(2-(2-(4-((9,10-bis(3,5-dimethylbenzyloxy)-2-hydroxy-4-methyl-1,3,4,6,7,11 b -hexahydro-2-hydro-pyrido[2,1-a]isoquinoline-2-yl)methyl)-1-hydro-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxo-piperidin-3-yl)isoindole-1,3-dione; Rac- (2 S 4 S ,11 bR )- N -(6-(4-((9,10-bis(3,5-dimethylbenzyloxy)-2-hydroxy-4-methyl-1,3,4,6,7,11 b -hexahydro-2-hydro-pyrido[2,1-a]isoquinoline-2-yl)methyl)-1-hydro-1,2,3-triazol-1-yl)n-hexyl)-2-((2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)acetamide; Rac- (2 S 4 S ,11 bR )-N-(2-(2-(2-(4-((9,10-bis(3,5-dimethylbenzyloxy)-2-hydroxy-4-methyl-1,3,4,6,7,11 b -hexahydro-2-hydro-pyrido[2,1-a]isoquinoline-2-yl)methyl)-1-hydro-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)-2-((2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)acetamide; Rac- (2 S 4 S ,11 bR )-4-((6-(4-((9,10-bis(3,5-dimethylbenzyloxy)-2-hydroxy-4-methyl-1,3,4,6,7,11 b -hexahydro-2-hydro-pyrido[2,1-a]isoquinoline-2-yl)methyl)-1-hydro-1,2,3-triazol-1-yl)n-hexyl)oxy)-2-(2,6-dioxo-piperidin-3-yl)isoindole-1,3-dione; Rac- (2 S 4 S ,11 bR )-5-((6-(4-((9,10-bis(3,5-dimethylbenzyloxy)-2-hydroxy-4-methyl-1,3,4,6,7,11 b -hexahydro-2-hydro-pyrido[2,1-a]isoquinoline-2-yl)methyl)-1-hydro-1,2,3-triazol-1-yl)n-hexyl)oxy)-2-(2,6-dioxo-piperidin-3-yl)isoindole-1,3-dione; Rac- (2 S 4 S ,11 bR )-4-(2-(2-(2-(4-((9,10-bis(3,5-dimethylbenzyloxy)-2-hydroxy-4-methyl-1,3,4,6,7,11 b -hexahydro-2-hydro-pyrido[2,1-a]isoquinoline-2-yl)methyl)-1-hydro-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethoxy)-2-(2,6-dioxo-piperidin-3-yl)isoindole-1,3-dione; Rac- (2 S 4 S ,11 bR )-5-(2-(2-(2-(4-((9,10-bis(3,5-dimethylbenzyloxy)-2-hydroxy-4-methyl-1,3,4,6,7,11 b -hexahydro-2-hydro-pyrido[2,1-a]isoquinoline-2-yl)methyl)-1-hydro-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethoxy)-2-(2,6-dioxo-piperidin-3-yl)isoindole-1,3-dione; ( R )-2-((2 S ,3 R )-3-amino-2-hydroxy-4-phenylbutyramide)- N -(6-(4-(( Rac- (2 S 4 S ,11b R )-9,10-bis(3,5-dimethylbenzyloxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2-hydro-pyrido[2,1-a]isoquinoline-2-yl)methyl)-1 H -1,2,3-triazol-1-yl)n-hexyl)-4-methylpentanamide; ( R )-2-((2 S ,3 R )-3-amino-2-hydroxy-4-phenylbutyramide)- N -(2-(2-(2-(4-(((2) S 4 S ,11b R )-9,10-bis(3,5-dimethylbenzyloxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2-hydro-pyrido[2,1-a]isoquinoline-2-yl)methyl)-1 H -1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)-4-methylpentanamide; (2 S 4 R )-1-(( S )-2-(6-(4-(( Rac -(2 S 4 S ,11b R )-9,10-bis(3,5-dimethylbenzyloxy)-2-hydroxy-4-methyl-1,3,4,6,7,11 b -hexahydro-2-hydro-pyridino[2,1-a]isoquinoline-2-yl)methyl)-1 H -1,2,3-triazol-1-yl)hexanoyl)-3,3-dimethylbutyryl)-4-hydroxy- N -(4-(4-methylthiazo-5-yl)benzyl)pyrrolidine-2-carboxamide; (2 S 4 R )-1-(( S )-14-(4-(( Rac -(2 S 4 S ,11b R )-9,10-bis(3,5-dimethylbenzyloxy)-2-hydroxy-4-methyl-1,3,4,6,7,11b-hexahydro-2-hydro-pyrido[2,1-a]isoquinoline-2-yl)methyl)-1 H -1,2,3-triazol-1-yl)-2-(tert-butyl)-4-oxo-6,9,12-trioxo-3-tetradecanoamide)-4-hydroxy- N -(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide.

2. A pharmaceutical preparation, characterized in that, This includes the benzoquinol alkaloid compound of claim 1 or a pharmaceutically acceptable salt thereof.

3. The pharmaceutical preparation as described in claim 2, characterized in that, The pharmaceutically acceptable salts include salts formed by the above-mentioned compounds with inorganic or organic acids, wherein the inorganic acid is selected from hydrochloric acid, sulfuric acid, nitric acid, or hydrobromic acid, and the organic acid is selected from methanesulfonic acid, toluenesulfonic acid, or trifluoroacetic acid.

4. The pharmaceutical preparation as described in claim 2, characterized in that, The dosage form of the pharmaceutical preparation is tablets, capsules, lozenges, pills, granules, powders, or aerosols.

5. The pharmaceutical preparation as described in claim 2, characterized in that, The dosage form of the pharmaceutical preparation is sugar-coated pills, solutions, emulsions, suspensions, or gels.

6. The pharmaceutical preparation as described in claim 2, characterized in that, The dosage form of the pharmaceutical preparation is a syrup.

7. The pharmaceutical preparation as described in claim 2, characterized in that, The pharmaceutical preparation also includes pharmaceutical excipients.

8. The use of a benzoquinol alkaloid compound of claim 1 or a pharmaceutical preparation of any one of claims 2-5 in the preparation of a GDI2 protein degrading agent.

9. The use of a benzoquinolone alkaloid compound according to claim 1 or a pharmaceutical preparation according to any one of claims 2-5 in the preparation of an antitumor drug, characterized in that, The antitumor drug is an anti-pancreatic cancer drug.

10. A biotinylated benzoquinoloxetine alkaloid probe, characterized in that, Its chemical structural formula is shown below: ； The probe is capable of identifying a molecular target, namely the GDI2 protein.

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